bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633
Authors: Klein C , Entian K D
Published: January 2020

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. Recently, we reported regarding genes spaB, spaT, and spaC (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992) which are involved in the biosynthesis of subtilin, and genes spaR and spaK (C. Klein, C. Kaletta, and K.-D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), which regulate subtilin biosynthesis via a histidine kinase/response regulator system. Further sequence analysis revealed the presence of three additional open reading frames, spaI, spaF, and spaG, downstream of the structural gene spaS. The spaI gene encodes a hydrophilic 19.3-kDa lipoprotein containing a consensus signal sequence, indicating that this protein might be membrane anchored. A similar gene, nisI, has been identified in the nisin producer. SpaF shows strong homology to members of the family of ABC transporters. spaG encodes a hydrophobic protein which might form the active transporter together with SpaF. Gene disruption mutants in all three genes were still able to produce subtilin; however, these mutants were more sensitive to subtilin than the wild-type strain. These results show that these genes are involved in the immunity mechanism of the producer strain. A similar involvement of an ABC transporter in the self-protection mechanism has been described for the McbE and McbF transporter, which confers immunity against microcin B17 in Escherichia coli. Mutants containing mutations in the genes spaR and spaK, which are responsible for regulation of subtilin biosynthesis, also became more sensitive to subtilin.(ABSTRACT TRUNCATED AT 250 WORDS)
Transposition in Lactobacillus sake and its abolition of lactocin S production by insertion of IS1163, a new member of the IS3 family
Authors: Skaugen M , Nes I F
Published: January 2020

This report presents the nucleotide sequence and insertional activity of IS1163, which is a new member of the IS3 family of transposable elements. Analysis of spontaneous mutants of the lactocin S-producing Lactobacillus sake strain L45 show that the bacteriocin-negative phenotype is due to either loss of the producer plasmid or the insertion of IS1163 into the lactocin S operon (las operon). The data further show that insertional inactivation of the lactocin S operon is the result of a transposition event involving a chromosomally located donor copy of IS1163. Although the insertions described are clustered within a 250-bp region of the las operon, there are no features of the insertion sites to suggest target-specific insertion of IS1163. The overlapping, frameshifted organization of the two major open reading frames found in IS1163 is typical for the IS3 family, but the structure of the putative frameshift region includes features which distinguish IS1163 from the other members of the group. The insertional activity of IS1163 in L. sake L45 has aided in identifying regions of pCIM1 essential for lactocin S production and may have further practical applications as a mutational tool in L. sake.
Cometabolic oxidation of polychlorinated biphenyls in soil with a surfactant-based field application vector
Authors: Lajoie C A , Layton A C , Sayler G S
Published: January 2020

Polychlorinated biphenyl (PCB)-degradative genes, under the control of a constitutive promoter, were cloned into a broad-host-range plasmid and a transposon. These constructs were inserted into a surfactant-utilizing strain, Pseudomonas putida IPL5, to create a field application vector (FAV) in which a surfactant-degrading organism cometabolizes PCB. By utilizing a surfactant not readily available to indigenous populations and a constitutive promoter, selective growth and PCB-degradative gene expression are decoupled from biphenyl. Since PCB degradation via the biphenyl degradation pathway is nonadaptive in the absence of biphenyl, there is no selective pressure for PCB gene maintenance. The recombinant strains exhibited degradative activity against 25 of 33 PCB congeners in Aroclor 1248 in the absence of biphenyl. Whole-cell enzyme assays indicated that PCB-degradative activity of a recombinant strain carrying the PCB genes on a plasmid was approximately twice that of the same strain carrying the PCB genes on a transposon. Plasmid loss rates in the absence of antibiotic selection averaged 7.4% per cell division and were highly variable between experiments. Surfactant-amended slurries of PCB-contaminated electric power plant substation soil were inoculated with approximately 10(5) recombinant cells per ml. The populations of the added strains increased to greater than 10(9) cells per ml in 2 days, and cell growth coincided with PCB degradation. By 15 days, 50 to 60% of the indicator congener 2,3,2',5'-tetrachlorobiphenyl was degraded. The effectiveness of PCB degradation by the plasmid-containing strain depended on plasmid stability. The transposon-encoded PCB genes were much more stable, and in surfactant-amended soil slurries, PCB degradation was more consistent between experiments.
Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth
Authors: Winding Anne , Binnerup Svend Jørgen , Sørensen Jan
Published: January 2020

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.
Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product
Authors: Reis M , Eschbach-Bludau M , Iglesias-Wind M I , Kupke T , Sahl H G
Published: January 2020

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Pep5 production and producer immunity are associated with the 20-kb plasmid pED503. A 1.3-kb KpnI fragment of pED503, containing the Pep5 structural gene pepA, was subcloned into the Escherichia coli-Staphylococcus shuttle vector pCU1, and the recombinant plasmid pMR2 was transferred to the Pep5- and immunity-negative mutant S. epidermidis 5 Pep5- (devoid of pED503). This clone did not produce active Pep5 but showed the same degree of insensitivity towards Pep5 as did the wild-type strain. Sequencing of the 1.3-kb KpnI-fragment and analysis of mutants demonstrated the involvement of two genes in Pep5 immunity, the structural gene pepA itself and pepI, a short open reading frame upstream of pepA. To identify the 69-amino-acid pepI gene product, we constructed an E. coli maltose-binding protein-PepI fusion clone. The immunity peptide PepI was detected in the soluble and membrane fractions of the wild-type strain and the immune mutants (harboring the plasmids pMR2 and pMR11) by immunoblotting with anti-maltose-binding protein-PepI antiserum. Strains harboring either pepI without pepA or pepI with incomplete pepA were not immune and did not produce PepI. Washing the membrane with salts and EDTA reduced the amount of PepI in this fraction, and treatment with Triton X-100 almost completely removed the peptide. Furthermore, PepI was hydrolyzed by proteases added to osmotically stabilized protoplasts. This suggests that PepI is loosely attached to the outside of the cytoplasmic membrane. Proline uptake and efflux experiments with immune and nonimmune strains also indicated that PepI may act at the membrane site.
Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine
Authors: Ullrich M. , Guenzi A. C. , Mitchell R. E. , Bender C. L.
Published: January 2020

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by
Pseudomonas syringae
pv. glycinea PG4180. Tn
5
mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn
5
insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of
P. syringae
pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into
P. syringae
pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by
P. syringae
pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of
P. syringae
pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into
P. syringae
pv. syringae B1; however, these two constructs did not enable
Escherichia coli
K-12 to synthesize CMA. The production of CMA in
P. syringae
strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.
Removal of Adsorbed Toxin Fragments That Modify
Bacillus thuringiensis
CryIC δ-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation
Authors: Luo Ke , Adang Michael J.
Published: January 2020

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from
Bacillus thuringiensis
insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against
Spodoptera exigua.
Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of
B. thuringiensis
CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to
S. exigua
larvae.
125
I-labeled CryIC bound with high affinity to brush border membrane vesicles from
S. exigua
larvae.
Cloning of a phenazine biosynthetic locus of Pseudomonas aureofaciens PGS12 and analysis of its expression in vitro with the ice nucleation reporter gene
Authors: Georgakopoulos D G , Hendson M , Panopoulos N J , Schroth M N
Published: January 2020

Pseudomonas aureofaciens PGS12 produces three phenazine antibiotics, in addition to siderophores, hydrogen cyanide, pyrrolnitrin, and indoleacetic acid. Tn5-259.7 transposon mutagenesis was carried out to identify and clone a chromosomal locus involved in phenazine biosynthesis. Three classes of mutants were obtained: mutants deficient in phenazine production (Phz-), mutants deficient in hydrogen cyanide production (HCN-), and mutants deficient in the production of both compounds. EcoRI DNA fragments that contained the transposon and flanking regions were cloned from three mutants with single-transposon insertions, one from each phenotypic class. Phenazine and hydrogen cyanide production was restored by complementation of Phz- or HCN- mutants with selected cosmids from a PGS12 genomic library. No cosmids that complemented the doubly deficient Phz-HCN- mutant were obtained. A promoterless ice nucleation reporter gene was inserted in a phenazine biosynthetic locus by Tn3-spice transposon mutagenesis of a cosmid which complemented a phenazine-minus mutant. Reporter gene fusions that expressed the ice nucleation phenotype and no longer complemented phenazine production were introduced into the PGS12 chromosome by marker exchange. The expression of this locus was then monitored under different culture conditions. Expression decreased at pH levels below 7, and it was not affected by iron. Shikimic acid and phenylalanine favored higher expression levels. Expression was reduced in media with low substrate concentrations, indicating the importance of nutrient availability.
Factors Influencing the Synthesis of Polysaccharide by
Bradyrhizobium japonicum
Bacteroids in Field-Grown Soybean Nodules
Authors: Streeter John G. , Salminen Seppo O. , Beuerlein James E. , Schmidt Walter H.
Published: January 2020

Certain strains of
Bradyrhizobium japonicum
produce large quantities of polysaccharide in soybean (
Glycine max
(L.) Merr.) nodules, and nodule polysaccharide (NPS) is different from that produced in culture. A previous survey of field-grown plants showed highly variable levels of NPS among field sites. To obtain clues about the possible function of NPS, we conducted two additional surveys of field-grown plants. The amount of polysaccharide in bulk samples of nodules was not associated with soil type, texture, slope, drainage, or any of the measured soil chemical properties except pH and [Ca]. Correlations with pH and [Ca] were positive and highly significant for two independent surveys involving a total of 77 sites in two years. In a preliminary comparison of high and low levels of Ca supplied to soybean plants grown in silica sand in a greenhouse, a high level of Ca (200 mg of Ca liter
-1
) increased the NPS level and increased the Ca content of the polysaccharide fraction.
B. japonicum
isolates from 450 nodules collected at 10 field sites in 1993 were used to form nodules on soybean plants grown in sand culture in a greenhouse in order to examine bacterial phenotype under controlled conditions. Results showed that the NPS level in the bulk nodule sample from any given site was a function of the proportion of nodule occupants that were capable of NPS synthesis. Thus, a higher soil pH and/or [Ca] may positively influence the survival of
B. japonicum
capable of synthesis of the nodule-specific polysaccharide.
Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of
Pseudomonas fluorescens
DF57 and
Pseudomonas putida
DF14
Authors: Kragelund Lene , Nybroe Ole
Published: January 2020

Changes in culturability and outer membrane protein profiles were investigated in
Pseudomonas fluorescens
DF57 and
Pseudomonas putida
DF14 during starvation for carbon, nitrogen, and phosphorus.
P. fluorescens
DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells.
P. putida
DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional
P. fluorescens
and
P. putida
strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.
Effect of
Penicillium chrysogenum
on Lignin Transformation
Authors: Rodríguez A. , Carnicero A. , Perestelo F. , de la Fuente G. , Milstein O. , Falcón M. A.
Published: January 2020

A strain of
Penicillium chrysogenum
has been isolated from pine forest soils in Tenerife (Canary Islands). This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particular cinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) and low levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, and synthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraft lignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry and klason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remaining in cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and 94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% of total applied radioactivity of O
14
CH
3
-organosolv lignin was recovered as
14
CO
2
after 30 days of incubation, and 18.5% of radioactivity from insoluble O
14
CH
3
-organosolv lignin was solubilized. After 26 days of incubation, 2.9% of
14
C-β-dehydrogenative polymerisate and 4.1% of
14
C-ring-dehydrogenative polymerisate evolved as
14
CO
2
.
Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D
Authors: Coon S L , Kotob S , Jarvis B B , Wang S , Fuqua W C , Weiner R M
Published: January 2020

Shewanella colwelliana D is a marine procaryote which produces a diffusible brown pigment that correlates with melA gene expression. Previously, melA had been cloned, sequenced, and expressed in Escherichia coli; however, the reaction product of MelA had not been identified. This report identifies that product as homogentisic acid, provides evidence that the pigment is homogentisic acid-melanin (pyomelanin), and suggests that MelA is p-hydroxyphenylpyruvate hydroxylase. This is the first report of pyomelanin in an obligate marine bacterium.
Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3
Authors: Oremland Ronald S. , Blum Jodi Switzer , Culbertson Charles W. , Visscher Pieter T. , Miller Laurence G. , Dowdle Phillip , Strohmaier Frances E.
Published: January 2020

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO
2
with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se
0
). Hence, SES-3 can carry out a complete reduction of selenate to Se
0
. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [
14
C]lactate to
14
CO
2
coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide
m
-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se
0
by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [
75
Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se
0
. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se
0
may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.
Azoreductase activity in bacteria associated with the greening of instant chocolate puddings
Authors: Dykes G A , Timm R G , von Holy A
Published: January 2020

Pseudomonas sp. strain AZR1 and Klebsiella sp. strain AZR2 were isolated from reconstituted instant chocolate puddings that had turned green and were found to have azoreductase activity. This activity was inducible and NADH dependent. Differences in dye reduction rates between the two strains were apparent, and substrate specificity related to dye structure was observed.
Oxidation of methyl fluoride and dimethyl ether by ammonia monooxygenase in Nitrosomonas europaea
Authors: Hyman M R , Page C L , Arp D J
Published: January 2020

Methyl fluoride and dimethyl ether were previously identified as inhibitors of ammonia oxidation and N2O production in autotrophic nitrifying bacteria. We demonstrate that methyl fluoride and dimethyl ether are substrates for ammonia monooxygenase and are converted to formaldehyde and a mixture of methanol and formaldehyde, respectively.
Effects of Ionic Strength on Bacterial Adhesion and Stability of Flocs in a Wastewater Activated Sludge System
Authors: Zita Anna , Hermansson Malte
Published: January 2020

The success of biological wastewater treatment is to a large extent governed by the ability of bacteria to induce floc formation, thereby facilitating the separation of particles from the treated water. We performed studies on the dynamics of floc stability, the desorption of cells from the flocs, and the reflocculation of detached material. The floc stability was affected by the ionic strength of the medium in a way that strongly suggests that the interactions between the floc components can be explained by the theory of Derjaugin, Landau, Verwey, and Overbeek (DLVO theory). At increasing concentrations of electrolytes, the stability of the flocs increased. However, above an ionic strength of about 0.1 the floc stability decreased, and it seems that at this high electrolyte concentration the DLVO theory cannot be applied. The reversibility of the electrostatic double-layer effects was experimentally shown by treating the sludge repeatedly with a low-ionic-strength solution until parts of the flocs detached. When salt was added at this point, flocs re-form, resulting in a dramatic decrease in the turbidity of the supernatant liquid. Both reflocculation and detachment of floc material were seen with calcium as well as with potassium. This finding clearly indicates that the reflocculation and destabilization of flocs were due to changes in double-layer thickness rather than bridging effects of multivalent ions such as calcium. The results indicate that the ionic strength may well be an important factor for the floc stability in wastewater in situ.
Inoculation of
Scytalidium thermophilum
in Button Mushroom Compost and Its Effect on Yield
Authors: Straatsma Gerben , Olijnsma Tineke W. , Gerrits Jan P. G. , Amsing Jos G. M. , Op Den Camp Huub J. M. , Van Griensven Leo J. L. D.
Published: January 2020

Scytalidium thermophilum
isolates in culture, as well as the endogenous strain(s) in mushroom compost, were inactivated at 70°C. This temperature was used to pasteurize composts for experiments. Of nine thermophilic fungal species, only
S. thermophilum
and
Myriococcum thermophilum
grew well on pasteurized compost in test tubes. The effect of both species on the crop yield of
Agaricus bisporus
mushrooms was studied. In solid-state fermentation rooms called tunnels, compost was pasteurized and inoculated. After incubation, the inoculated organisms were reisolated and counted, showing their successful colonization. The yield of mushrooms on inoculated composts was almost twice that on the pasteurized control. This result demonstrates the effectiveness of
S. thermophilum
in compost preparation. Inoculation is not necessary for traditional compost preparation. Naturally occurring strains of
S. thermophilum
, present in ingredients, readily colonize compost during preparation. Inoculation may be vital if compost is pretreated at a high temperature in tunnels. This finding is of relevance for the environmentally controlled production of high-yielding compost.
Simulation Model of the Coupling between Nitrification and Denitrification in a Freshwater Sediment
Authors: Blackburn T. Henry , Blackburn Nicholas D. , Jensen Kim , Risgaard-Petersen Nils
Published: January 2020

A model was constructed to simulate the results of experiments which investigated nitrification and denitrification in the freshwater sediment of Lake Vilhelmsborg, Denmark (K. Jensen, N. P. Sloth, N. Risgaard-Petersen, S. Rysgaard, and N. P. Revsbech, Appl. Environ. Microbiol. 60:2094-2100, 1994). The model output faithfully represented the profiles of O
2
and NO
3
-
and rates of nitrification, denitrification, and O
2
consumption as the O
2
concentration in the overlying water was increased from 10 to 600 μM. The model also accurately predicted the response, to increasing O
2
concentrations, of the integrated (micromoles per square meter per hour) rates of nitrification and denitrification. The simulated rates of denitrification of NO
3
-
diffusing from the overlying water (

D
w

) and of NO
3
-
generated by nitrification within the sediment (

D
n

) corresponded to the experimental rates as the O
2
concentration in the overlying water was altered. The predicted

D
w

and

D
n

rates, as NO
3
-
concentration in the overlying water was changed, closely resembled those determined experimentally. The model was composed of 41 layers 0.1 mm thick, of which 3 represented the diffusive boundary layer in the water. Large first-order rate constants for nitrification and denitrification were required to completely oxidize all NH
4
+
diffusing from the lower sediment layers and to remove much of the NO
3
-
produced. In addition to the flux of NH
4
+
from below, the model required a flux of an electron donor, possibly methane. Close coupling between nitrification and denitrification, achieved by allowing denitrification to tolerate some O
2
(∼10 μM), was necessary to reproduce the real data. Spatial separation of the two processes (no toleration by denitrification of O
2
) resulted in too high NO
3
-
concentrations and too low rates of denitrification.
Minerals Associated with Biofilms Occurring on Exposed Rock in a Granitic Underground Research Laboratory
Authors: Brown D. Ann , Kamineni D. Choudari , Sawicki Jerzy A. , Beveridge Terry J.
Published: January 2020

The concept of disposal of nuclear fuel waste in crystalline rock requires the effects of microbial action to be investigated. The Underground Research Laboratory excavated in a pluton of the Canadian Shield provides a unique opportunity to study these effects. Three biofilms kept moist by seepage through fractures in granitic rock faces of the Underground Research Laboratory have been examined. The biofilms contained a variety of gram-negative and gram-positive morphotypes held together by an organic extracellular matrix. Nutrient levels in the groundwater were low, but energy-dispersive X-ray spectroscopy has shown biogeochemical immobilization of several elements in the biofilms; some of these elements were concentrated from extremely dilute environmental concentrations, and all elements were chemically complexed together to form amorphous or crystalline fine-grained minerals. These were seen by transmission electron microscopy to be both associated with the surfaces of the bacteria and scattered throughout the extracellular matrix, suggesting their de novo development through bacterial surface-mediated nucleation. The biofilm consortia are thought to concentrate elements both by passive sorption and by energy metabolism. By Mössbauer spectroscopy and X-ray diffraction, one of the biofilms showed that iron was both oxidized and precipitated as ferrihydrite or hematite aerobically and reduced and precipitated as siderite anaerobically. We believe that some Archean banded-iron formations could have been formed in a manner similar to this, as it would explain the deposition of hematite and siderite in close proximity. This biogeochemical development of minerals may also affect the transport of material in waste disposal sites.
Microscopic and Thermal Characterization of Hydrogen Peroxide Killing and Lysis of Spores and Protection by Transition Metal Ions, Chelators, and Antioxidants
Authors: Shin Soon-Young , Calvisi Elizabeth G. , Beaman Teofila C. , Pankratz H. Stuart , Gerhardt Philipp , Marquis Robert E.
Published: January 2020

Killing of bacterial spores by H
2
O
2
at elevated but sublethal temperatures and neutral pH occurred without lysis. However, with prolonged exposure or higher concentrations of the agent, secondary lytic processes caused major damage successively to the coat, cortex, and protoplast, as evidenced by electron and phase contrast microscopy. These processes were also reflected in changes in differential scanning calorimetric profiles for H
2
O
2
-treated spores. Endothermic transitions in the profiles occurred at lower temperatures than usual as a result of H
2
O
2
damage. Thus, H
2
O
2
sensitized the cells to heat damage. Longer exposure to H
2
O
2
resulted in total disappearance of the transitions, indicative of major disruptions of cell structure. Spores but not vegetative cells were protected against the lethal action of H
2
O
2
by the transition metal cations Cu
+
, Cu
2+
, Co
2+
, Co
3+
, Fe
2+
, Fe
3+
, Mn
2+
, Ti
3+
, and Ti
4+
. The metal chelator EDTA was also somewhat protective, while
o
-phenanthroline, citrate, deferoxamine, and ethanehydroxydiphosphonate were only marginally so. Superoxide dismutase and a variety of other free-radical scavengers were not protective. In contrast, reducing agents such as sulfhydryl compounds and ascorbate at concentrations of 20 to 50 mM were highly protective. Decoating or demineralization of the spores had only minor effects. The marked dependence of H
2
O
2
sporicidal activity on moderately elevated temperature and the known low reactivity of H
2
O
2
itself suggest that radicals are involved in its killing action. However, the protective effects of a variety of oxidized or reduced transition metal ions indicate that H
2
O
2
killing of spores is markedly different from that of vegetative cells.
Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat
Authors: Degnan A J , Kaspar C W , Otwell W S , Tamplin M L , Luchansky J B
Published: January 2020

Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat
Authors: Daugherty D D , Karel S F
Published: January 2020

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)
Relationship between Phylogeny and Pathotype for the Bacterial Blight Pathogen of Rice
Authors: Nelson Rebecca J. , Baraoidan Marietta R. , Cruz Casiana M. Vera , Yap Immanuel V. , Leach Jan E. , Mew Twng W. , Leung Hei
Published: January 2020

Several transposable elements were isolated from the genome of
Xanthomonas oryzae
pv. oryzae. These elements and an avirulence gene isolated from
X. oryzae
pv. oryzae were used as hybridization probes for a collection of
X. oryzae
pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races of
X. oryzae
pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different phenetic groups were inoculated on rice cultivars carrying 10 resistance genes. Two new races were differentiated, corresponding to pathogen lineages identified by DNA typing. On the basis of DNA and pathotypic analyses, together with information on the spatial and temporal distribution of the pathogen types from this and other studies, a general picture of
X. oryzae
pv. oryzae evolution in the Philippines is presented.
Changes in Ester-Linked Phospholipid Fatty Acid Profiles of Subsurface Bacteria during Starvation and Desiccation in a Porous Medium
Authors: Kieft T. L. , Ringelberg D. B. , White D. C.
Published: January 2020

Ester-linked phospholipid fatty acid (PLFA) profiles of a
Pseudomonas aureofaciens
strain and an
Arthrobacter protophormiae
strain, each isolated from a subsurface sediment, were quantified in a starvation experiment in a silica sand porous medium under moist and dry conditions. Washed cells were added to sand microcosms and maintained under saturated conditions or subjected to desiccation by slow drying over a period of 16 days to final water potentials of approximately - 7.5 MPa for the
P. aureofaciens
and - 15 MPa for the
A. protophormiae.
In a third treatment, cells were added to saturated microcosms along with organic nutrients and maintained under saturated conditions. The numbers of culturable cells of both bacterial strains declined to below detection level within 16 days in both the moist and dried nutrient-deprived conditions, while direct counts and total PLFAs remained relatively constant. Both strains of bacteria maintained culturability in the nutrient-amended microcosms. The dried
P. aureofaciens
cells showed changes in PLFA profiles that are typically associated with stressed gram-negative cells, i.e., increased ratios of saturated to unsaturated fatty acids, increased ratios of
trans
- to
cis
-monoenoic fatty acids, and increased ratios of cyclopropyl fatty acids to their monoenoic precursors.
P. aureofaciens
starved under moist conditions showed few changes in PLFA profiles during the 16-day incubation, whereas cells incubated in the presence of nutrients showed decreases in the ratios of both saturated fatty acids to unsaturated fatty acids and cyclopropyl fatty acids to their monoenoic precursors. The PLFA profiles of
A. protophormiae
changed very little in response to either nutrient deprivation or desiccation. Diglyceride fatty acids, which have been proposed to be indicators of dead or lysed cells, remained relatively constant throughout the experiment. Only the
A. protophormiae
desiccated for 16 days showed an increase in the ratio of diglyceride fatty acids to PLFAs. The results of this laboratory experiment can be useful for interpreting PLFA profiles of subsurface communities of microorganisms for the purpose of determining their physiological status.
Effects of Methane Metabolism on Nitrification and Nitrous Oxide Production in Polluted Freshwater Sediment
Authors: Roy Réal , Knowles Roger
Published: January 2020

We report the effect of CH
4
and of CH
4
oxidation on nitrification in freshwater sediment from Hamilton Harbour, Ontario, Canada, a highly polluted ecosystem. Aerobic slurry experiments showed a high potential for aerobic N
2
O production in some sites. It was suppressed by C
2
H
2
, correlated to NO
3
-
production, and stimulated by NH
4
+
concentration, supporting the hypothesis of a nitrification-dependent source for this N
2
O production. Diluted sediment slurries supplemented with CH
4
(1 to 24 μM) showed earlier and enhanced nitrification and N
2
O production compared with unsupplemented slurries (≤1 μM CH
4
). This suggests that nitrification by methanotrophs may be significant in freshwater sediment under certain conditions. Suppression of nitrification was observed at CH
4
concentrations of 84 μM and greater, possibly through competition for O
2
between methanotrophs and NH
4
+
-oxidizing bacteria and/or competition for mineral N between these two groups of organisms. In Hamilton Harbour sediment, the very high CH
4
concentrations (1.02 to 6.83 mM) which exist would probably suppress nitrification and favor NH
4
+
accumulation in the pore water. Indeed, NH
4
+
concentrations in Hamilton Harbour sediment are higher than those found in other lakes. We conclude that the impact of CH
4
metabolism on N cycling processes in freshwater ecosystems should be given more attention.
Characterization of Inorganic Biocarriers That Moderate System Upsets during Fixed-Film Biotreatment Processes
Authors: Durham Don R. , Marshall Leda C. , Miller James G. , Chmurny Alan B.
Published: January 2020

Inorganic matrices were developed for fixed-film bioreactors affording protection to microorganisms and preventing loss of bioreactor productivity during system upsets. These biocarriers, designated Type-Z, contain ion-exchange properties and possess high porosity and a high level of surface area, which provide a suitable medium for microbial colonization. Viable cell populations of 10
9
/g were attainable, and scanning electron micrographs revealed extensive external colonization and moderate internal colonization with aerobic microorganisms. Laboratory-scale bioreactors were established with various biocarriers and colonized with
Pseudomonas aeruginosa
, and comparative studies were performed. The data indicated that bioreactors containing the Type-Z biocarriers were more proficient at removing phenol (1,000 ppm) than bioreactors established with Flexirings (plastic) and Celite R635 (diatomaceous earth) biocarriers. More significantly, these biocarriers were shown to moderate system upsets that affect operation of full-scale biotreatment processes. For example, subjecting the Type-Z bioreactor to an influent phenol feed at pH 2 for periods of 24 h did not decrease the effluent pH or reactor performance. In contrast, bioreactors containing either Celite or Flexirings demonstrated an effluent pH drop to ∼2.5 and a reduction in reactor performance by 75 to 82%. The Celite reactor recovered after 5 days, whereas the bioreactors containing Flexirings did not recover. Similar advantages were noted during either nutrient or oxygen deprivation experiments as well as alkali and organic system shocks. The available data suggest that Type-Z biocarriers represent an immobilization medium that provides an amenable environment for microbial growth and has the potential for improving the reliability of fixed-film biotreatment processes.
Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate
Authors: Landa A S , Sipkema E M , Weijma J , Beenackers A A , Dolfing J , Janssen D B
Published: January 2020

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.
Anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2 proceeds via carboxylation to protocatechuate
Authors: Gorny N , Schink B
Published: January 2020

Under anoxic conditions, most methoxylated mononuclear aromatic compounds are degraded by bacteria, with catechol being formed as an important intermediate. On the basis of our experiments with the sulfate-reducing bacterium Desulfobacterium sp. strain Cat2, we describe for the first time the enzymatic activities involved in the complete anaerobic oxidation of catechol and protocatechuate. Results obtained from experiments with dense cell suspensions of strain Cat2 demonstrated that all enzymes necessary for protocatechuate and benzoate degradation were induced during growth with catechol. In addition, anaerobic oxidation of catechol was found to be a CO2-dependent process. Phenol was not degraded in suspensions of cells grown with catechol. In cell extracts of Desulfobacterium sp. strain Cat2, protocatechuyl-coenzyme A (CoA) was formed from catechol, bicarbonate, and uncombined CoA. This oxygen-sensitive reaction requires high concentrations of both bicarbonate and protein, and only very low levels of enzyme were detected. In a second oxygen-sensitive step, protocatechuyl-CoA was reduced to 3-hydroxybenzoyl-CoA by reductive elimination of the p-hydroxyl group. Further dehydroxylation to benzoyl-CoA was not detectable. Key reactions described for anaerobic degradation of benzoate were catalyzed by cell extracts of strain Cat2, too.
Isolation of
Azospirillum lipoferum
4T Tn
5
Mutants Affected in Melanization and Laccase Activity
Authors: Faure D. , Bouillant M. L. , Bally R.
Published: January 2020

Azospirillum lipoferum
4T has original properties such as nonmotility, melanin synthesis, and laccase activity. Following random Tn
5
mutagenesis in
A. lipoferum
4T, we obtained 10 mutants which were affected in melanization and laccase activity. The class 1 mutants, with intermediate levels of laccase activity, showed some coloration; the class 2 mutants, which were completely negative for laccase activity, were also colorless. The Tn
5
localization on the chromosome or on the cryptic 300-MDa plasmid of
A. lipoferum
4T was proven by hybridization for all class 1 mutants or for most class 2 mutants, respectively.
Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method
Authors: Lantz P G , Tjerneld F , Borch E , Hahn-Hägerdal B , Rådström P
Published: January 2020

A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase.