bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic improvement of Escherichia coli for enhanced biological removal of phosphate from wastewater
Authors: Kato J , Yamada K , Muramatsu A , Hardoyo  , Ohtake H
Published: January 2020

The ability of Escherichia coli MV1184 to accumulate inorganic phosphate (Pi) was enhanced by manipulating the genes involved in the transport and metabolism of Pi. The high-level Pi accumulation was achieved by modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate-inducible transport system (pstS, pstC, pstA, and pstB). Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. Recombinant strains, which contained either pBC29 (carrying ppk) or pEP02.2 (pst operon), removed approximately two- and threefold, respectively, more Pi from minimal medium than did the control strain. The highest rates of Pii removal were obtained by strain MV1184 containing pEP03 (ppk and ackA). However, unlike the control strain, MV1184 (pEP03) released Pi to the medium after growth had stopped. Drastic changes in growth and Pi uptake were observed when pBC29 (ppk) and pEP02.2 (pst operon) were introduced simultaneously into MV1184. Even though growth of this recombinant was severely limited in minimal medium, the recombinant could remove approximately threefold more Pi than the control strain. Consequently, the phosphorus content of this recombinant reached a maximum of approximately 16% on a dry weight basis (49% as phosphate).
Involvement of cytochromes in the anaerobic biotransformation of tetrachloromethane by Shewanella putrefaciens 200
Authors: Picardal F W , Arnold R G , Couch H , Little A M , Smith M E
Published: January 2020

Shewanella putrefaciens 200 is an obligate respiratory bacterium that can utilize a variety of terminal electron acceptors, e.g., NO3-, NO2-, Fe(III), and trimethylamine N-oxide, in the absence of O2. The bacterium catalyzed the reductive transformation of tetrachloromethane (CT) under anaerobic conditions. The only identified product was trichloromethane (CF), but CF production was not stoichiometric. No dichloromethane, chloromethane, or methane was produced. A chloride mass balance indicated that fully dechlorinated products were not formed. Studies with [14C]CT suggested that a portion of the transformed CT reacted with biomass to form unidentified soluble and insoluble products. Intermediate production of a trichloromethyl radical can explain observed product distribution without significant CO2 formation. Evidence suggests that respiratory c-type cytochromes are responsible for the dehalogenation ability of S. putrefaciens 200. Previous growth under microaerobic conditions ([O2], < 2.5 microM) results in (i) a 2.6-fold increase in specific heme c content and (ii) a 2.3-fold increase in specific rates of anaerobic CT transformation. Manipulation of heme content by growth on iron-free medium or medium amended with delta-aminolevulinic acid showed that CT transformation rates increase with increases in specific heme c content. Transformation of CT is inhibited by CO. Dehalogenation studies with periplasmic, cytoplasmic, and membrane fractions indicated that only periplasmic and membrane fractions possessed dehalogenation ability. Cytochromes c were the predominant cytochromes present. Membranes were also found to contain smaller amounts of cytochrome b. Observed CT transformation patterns are consistent with a cometabolic description involving fortuitous CT reduction by reduced c-type cytochromes.
Isolation and Characterization of Nisin-Resistant
Leuconostoc mesenteroides
for Use in Cabbage Fermentations
Authors: Breidt F. , Crowley K. A. , Fleming H. P.
Published: January 2020

Leuconostoc mesenteroides
strains that are resistant to high levels of nisin (up to 25,000 IU/ml in broth) were isolated. These nisin-resistant mutants were evaluated to determine their potential use as starter culture strains for cabbage fermentations. We found that some
L. mesenteroides
strains could be adapted to high levels of nisin resistance, while others could not. The nisin resistance trait was found to be stable for at least 35 generations, in the absence of nisin selection, for all mutants tested. The effects of nisin and salt, separately and in combination, on growth kinetics of the nisin-resistant strains were determined. Salt was the most influential factor on the specific growth rates of the mutants, and no synergistic effect between nisin and salt on specific growth rates was observed. The nisin-resistant strains were unimpaired in their ability to rapidly produce normal heterolactic fermentation end products. The use of these
L. mesenteroides
mutants as starter cultures in combination with nisin may extend the heterolactic phase of cabbage fermentations.
Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene
Authors: Erickson B D , Mondello F J
Published: January 2020

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.
Secretion of streptavidin from Bacillus subtilis
Authors: Nagarajan V , Ramaley R , Albertson H , Chen M
Published: January 2020

Streptavidin is an extracellular tetrameric protein produced by Streptomyces avidinii. A series of hybrid gene fusions consisting of Bacillus signal peptide coding regions fused to the mature streptavidin sequence were constructed. B. subtilis strains harboring these plasmids accumulate a tetrameric streptavidin in the growth medium. The properties of the streptavidin produced by B. subtilis are similar to those of the streptavidin produced by S. avidinii. B. subtilis strains carrying the various fusions can be grown to a high cell density in a biotin-free medium. Thus, B. subtilis represents an alternate host system for the production of streptavidin.
Molecular analysis of the lactacin F operon
Authors: Fremaux C , Ahn C , Klaenhammer T R
Published: January 2020

Lactacin F is a nonlantibiotic, heat-stable, peptide bacteriocin produced by Lactobacillus johnsonii VPI11088. Molecular analysis of the lactacin F DNA region characterized a small operon that codes for three open reading frames, designated lafA, lafX, and ORFZ. The peptide encoded by lafA, the lactacin F structural gene, was compared with various peptide bacteriocins from lactic acid bacteria, and similarities were identified in the amino and carboxy termini of the propeptides. Site-directed mutagenesis of the LafA precursor at the two glycine residues in positions -1 and -2 defined an essential motif for processing of mature lactacin F. The involvement of the peptides encoded by lafX and ORFZ in bacteriocin expression was investigated by subcloning various fragments from the lactacin F region into the shuttle vector pGKV210. In addition to lafA, expression of lafX is essential to lactacin F activity. The lactacin F operon resembles the genetic organization of lactococcin M. Although no function has been assigned to ORFZ by genetic analysis, both peptide Z and the lactococcin M immunity protein are predicted to be integral membrane proteins with four putative transmembrane segments. Lactacin F activity, defined by bactericidal action on Lactobacillus delbrueckii, is dependent on the expression of two genes, lafA and lafX.
Bacterioplankton Growth Yield: Seasonal Variations and Coupling to Substrate Lability and β-Glucosidase Activity
Authors: Middelboe Mathias , Søndergaard Morten
Published: January 2020

The seasonal variation in the carbon growth yield of pelagic bacteria in the eutrophic lake Frederiksborg Slotssø was studied. The growth yield was determined in dilution culture experiments, in which a substrate of dissolved organic carbon (DOC) from the lake was incubated with a natural bacterioplankton assemblage. Bacterial growth efficiency varied annually from 8 to 60% with an average (and standard deviation) of 41 ± 11% (
n
= 29). Simultaneous measurements of growth yield, substrate lability (DOC
L
), chlorophyll and bacterial production, abundance, and extracellular enzymatic activity revealed new aspects of the regulation of bacterial DOC utilization. Growth yield correlated positively to DOC
L
and negatively to β-
d
-glucosidase activity. These results indicated a close coupling between the substrate conditions and the physiological response of the bacteria. The large variations in yield within a few days and the close coupling to substrate availability showed that one single global carbon yield factor cannot be expected to apply in pelagic systems.
Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination
Authors: Le Guyader F , Apaire-Marchais V , Brillet J , Billaudel S
Published: January 2020

Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts).
Effect of Phosphate Concentration on Production of Tetrodotoxin by
Alteromonas tetraodonis
Authors: Gallacher Susan , Birkbeck T. H.
Published: January 2020

Tetrodotoxin production by
Alteromonas tetraodonis
occurred during the stationary phase of growth and was regulated by phosphate concentration; toxin production was repressed if phosphate was added at the onset of stationary phase and was over 100-fold greater in phosphate-limited cultures than in cultures in which phosphate was not limiting.
Measurements of Seasonal Rates and Annual Budgets of Organic Carbon Fluxes in an Antarctic Coastal Environment at Signy Island, South Orkney Islands, Suggest a Broad Balance between Production and Decomposition
Authors: Nedwell D. B. , Walker T. R. , Ellis-Evans J. C. , Clarke A.
Published: January 2020

We report here the first comprehensive seasonal study of benthic microbial activity in an Antarctic coastal environment. Measurements were made from December 1990 to February 1992 of oxygen uptake and sulfate reduction by inshore coastal sediments at Signy Island, South Orkney Islands, Antarctica. From these measurements the rate of benthic mineralization of organic matter was calculated. In addition, both the deposition rate of organic matter to the bottom sediment and the organic carbon content of the bottom sediment were measured during the same period. Organic matter input to the sediment was small under winter ice cover, and the benthic respiratory activity and the organic content of the surface sediment declined during this period as available organic matter was depleted. On an annual basis, about 32% of benthic organic matter mineralization was anoxic, but the proportion of anoxic compared with oxic mineralization increased during the winter as organic matter was increasingly buried by the amphipod infauna. Fresh organic input occurred as the sea ice melted and ice algae biomass sedimented onto the bottom, and input was sustained during the spring after ice breakup by continued primary production in the water column. The benthic respiratory rate and benthic organic matter content correspondingly increased towards the end of winter with the input of this fresh organic matter. The rates of oxygen uptake during the southern summer (80 to 90 mmol of O
2
m
-2
day
-1
) were as high as those reported for other sediments at much higher environmental temperatures, and the annual mineralization of organic matter was equally high (12 mol of C m
-2
year
-1
). Seasonal variations of benthic activity in this antarctic coastal sediment were regulated by the input and availability of organic matter and not by seasonal water temperature, which was relatively constant at between -1.8 and 0.5°C. We conclude that despite the low environmental temperature, organic matter degradation broadly balanced organic matter production, although there may be significant interrannual variations in the sources of the organic matter inputs.
Surface Chemistry of
Thiobacillus ferrooxidans
Relevant to Adhesion on Mineral Surfaces
Authors: Devasia Preston , Natarajan K. A. , Sathyanarayana D. N. , Rao G. Ramananda
Published: January 2020

Thiobacillus ferrooxidans
cells grown on sulfur, pyrite, and chalcopyrite exhibit greater hydrophobicity than ferrous ion-grown cells. The isoelectric points of sulfur-, pyrite-, and chalcopyrite-grown cells were observed to be at a pH higher than that for ferrous ion-grown cells. Microbe-mineral interactions result in change in the surface chemistry of the organism as well as that of the minerals with which it has interacted. Sulfur, pyrite, and chalcopyrite after interaction with
T. ferrooxidans
exhibited a significant shift in their isoelectric points from the initial values exhibited by uninteracted minerals. With antibodies raised against sulfur-grown
T. ferrooxidans
, pyrite- and chalcopyrite-grown cells showed immunoreactivity, whereas ferrous ion-grown cells failed to do so. Fourier transform infrared spectroscopy of sulfur-grown cells suggested that a proteinaceous new cell surface appendage synthesized in mineral-grown cells brings about adhesion to the solid mineral substrates. Such an appendage was found to be absent in ferrous ion-grown cells as it is not required during growth in liquid substrates.
Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction
Authors: Oyofo B A , Rollins D M
Published: January 2020

A previously developed polymerase chain reaction (PCR) amplification of a target region in the flaA Campylobacter flagellin gene was evaluated and adapted for use with environmental water samples. The ability to detect Campylobacter jejuni or Campylobacter coli in seeded water samples was tested with various filters after concentration and freeze-thaw lysis of the bacterial cells. A nonradioactive probe for the amplified flagellin gene fragment detected as little as 1 to 10 fg of genomic DNA and as few as 10 to 100 viable C. jejuni cells per 100 ml of water filtered onto Fluoropore (Millipore Corp.) filters. No amplification was obtained with cellulose acetate filters, most likely because of binding of the DNA to the filter. Concentration and lysis of target cells on Fluoropore and Durapore (Millipore Corp.) filters allowed PCR to be performed in the same reaction tube without removing the filters. This methodology was then adapted for use with environmental water samples. The water supply to a broiler chicken production farm was suspected as the source of C. jejuni known to be endemic in grow-out flocks at the farm, despite the inability to culture the organisms by standard methods. The filtration-PCR method detected Campylobacter DNA in more than half of the farm water samples examined. Amplified campylobacter DNA was not detected in small volumes of regional surface water samples collected on a single occasion in February. The filtration-PCR amplification method provided a basis for detection of C. jejuni and C. coli in environmental waters with a high degree of specificity and sensitivity.
Cloning and Mutagenesis of a Cytochrome P-450 Locus from
Bradyrhizobium japonicum
That Is Expressed Anaerobically and Symbiotically
Authors: Tully Raymond E. , Keister Donald L.
Published: January 2020

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of
Bradyrhizobium japonicum.
Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of
B. japonicum
USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in
Escherichia coli
produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown
B. japonicum
cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the
CYP112
locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.
Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens
Authors: DiChristina T J , DeLong E F
Published: January 2020

A 16S rRNA-targeted oligonucleotide probe specific for the iron (Fe3+)- and manganese (Mn4+)-reducing bacterium Shewanella putrefaciens was constructed and tested in both laboratory- and field-based hybridization experiments. The radioactively labeled probe was used to detect S. putrefaciens in field samples collected from the water column and sediments of Oneida Lake in New York and its major southern tributary, Chittenango Creek. S. putrefaciens was quantified by (i) hybridization of the probe to bulk RNA extracted from field samples and normalization of the S. putrefaciens-specific rRNA to total eubacterial rRNA, (ii) a colony-based probe hybridization assay, and (iii) a colony-based biochemical assay which detected the formation of iron sulfide precipitates on triple-sugar iron agar. The results of field applications indicated that the three detection methods were comparable in sensitivity for detecting S. putrefaciens in water column and sediment samples. S. putrefaciens rRNA was detected in the surficial layers of the lake and creek sediments, but the levels of S. putrefaciens rRNA were below the detection limits in the lake and creek water samples. The highest concentrations of S. putrefaciens rRNA, corresponding to approximately 2% of the total eubacterial rRNA, were detected in the surficial sediments of Chittenango Creek and at a midlake site where the Oneida Lake floor is covered by a high concentration of ferromanganese nodules. This finding supports the hypothesis that metal-reducing bacteria such as S. putrefaciens are important components in the overall biogeochemical cycling of iron, manganese and other elements in seasonally anoxic freshwater basins.
Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent
Enterobacter cloacae
Authors: Loper Joyce E. , Ishimaru Carol A. , Carnegie Susan R. , Vanavichit Apichart
Published: January 2020

Five strains of
Enterobacter cloacae
that are biological control agents of
Pythium
damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain
E. cloacae
EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to
Escherichia coli
DH5α. The aerobactin biosynthesis genes of
E. cloacae
hybridized to those of the pColV-K30 plasmid of
E. coli
, but restriction patterns of the aerobactin regions of pColV-K30 and
E. cloacae
differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of
Pythium
damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.
Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils
Authors: Estrella M R , Brusseau M L , Maier R S , Pepper I L , Wierenga P J , Miller R M
Published: January 2020

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.
Identification of Molecular Variants in Mitochondrial DNAs of Members of the Genera
Beauveria, Verticillium, Paecilomyces, Tolypocladium
, and
Metarhizium
Authors: Hegedus Dwayne D. , Khachatourians George G.
Published: January 2020

A set of five mitochondrial (mt) probes derived from a strain of
Beauveria bassiana
was used to evaluate the similarity of mtDNAs from 15 additional isolates of this fungus and five genera of other entomopathogenic fungi. The probes and genes encoded for (shown in parentheses) were pBbmtE2 (
NADI, ATP6
), pBbmtE3 (
ATP6
, small rRNA [
srRNA
]), pBbmtE4 (
srRNA, CO3, NAD6
), pBbSE1 (

NAD6, tRNA
Val, Ile, Ser, Trp, Pro

, large rRNA [
lrRNA
]), and pBbXS1 (
lrRNA
). The probes produced identical hybridization patterns in
Eco
RI-digested DNA from nearly all isolates of
B. bassiana
and
Beauveria caledonica
. Similar patterns were also observed with
Beauveria densa
. The isolates of
B. caledonica
and
B. densa
DNAs could be differentiated from each other and from
B. bassiana
on the basis of a
Hin
dIII digestion and probing with pBbmtE3. Probe pBbmtE2 produced either a 5.0-kb or a 4.1-kb band in all of the
B. bassiana
isolates. This observation was used to categorize the mtDNA of
B. bassiana
into two types, designated A and B. Hybridization of the five probes produced distinct banding patterns in
Beauveria brongniartii, Tolypocladium cylindrosporum, Tolypocladium nivea, Metarhizium anisopliae, Verticillium lecanii
, and
Paecilomyces farinosus
. Hybridizations carried out with multiple probes simultaneously present produced unique patterns which characterized the
B. bassiana
group from all other fungi tested. These results are discussed in terms of how mtDNA polymorphisms in
B. bassiana
may relate to natural population structures, mt transmission in deuteromycetes, and the use of mtDNA polymorphisms in structural analysis of mtDNA.
Remobilization of Heavy Metals Retained as Oxyhydroxides or Silicates by
Bacillus subtilis
Cells
Authors: Urrutia Matilde M. , Beveridge Terry J.
Published: January 2020

The stability of heavy metals bound as either silicates or oxyhydroxides by the surfaces of
Bacillus subtilis
with changes in pH and presence of competing cations and EDTA was studied. Wall-bound silicate was very stable. Of all conditions, a pH of 3.0 (by HNO
3
) promoted the greatest release of Cd (87% [wt/wt]), Pb (77%), or Cu (54%). Bacterial Pb resisted extraction by EDTA, and Cd that was included in bacterial-silicates proved difficult to remobilize.
A Strategy for Rotation of Different Bacteriophage Defenses in a Lactococcal Single-Strain Starter Culture System
Authors: Sing Wesley D. , Klaenhammer Todd R.
Published: January 2020

A new strategy for starter culture rotations was developed for a series of phage-resistant clones genetically derived from a single strain of
Lactococcus lactis
subsp.
lactis.
Phage-resistant derivatives carrying different defense systems were constructed via conjugation with various plasmids encoding abortive infection (Abi/Hsp) and/or restriction and modification (R/M) systems of different specificity. The plasmids included pTR2030 (Hsp
+
R
+
/M
+
), pTN20 (Abi
+
R
+
/M
+
), pTRK11 (R
+
/M
+
), and pTRK68 (R
+
/M
+
). Selected phage-resistant transconjugants or transformants were evaluated in different rotation sequences through cycles of the Heap-Lawrence starter culture activity test in milk contaminated with phage and whey from the previous cycle. When used in consecutive sequence, derivative strains carrying the R/M systems encoded by pTN20, pTRK11, and pTRK68 retarded phage development when the initial levels of phage contamination were below 10
2
PFU/ml but not when levels were increased to 10
3
PFU/ml. Use of a derivative bearing pTR2030 (Hsp
+
R
+
/M
+
) at the beginning of the rotation prevented phage development, even when the initial levels of phage contamination were high (10
6
PFU/ml). Alternating the type and specificity of R/M and Abi defenses through the rotation prevented phage proliferation and in some cases eliminated contaminating phages. A model rotation sequence for the phage defense rotation strategy was developed and performed successfully over nine cycles of the Heap-Lawrence starter culture activity test in the presence of high-titer commercial phage composites. This phage defense rotation strategy is designed to protect a highly specialized
Lactococcus
strain from phage attack during continuous and extended use in the dairy industry.
Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium
Vibrio harveyi
to Chitin
Authors: Montgomery Michael T. , Kirchman David L.
Published: January 2020

We examined the mechanism of attachment of the marine bacterium
Vibrio harveyi
to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of
V. harveyi
to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of
V. harveyi
to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of
V. harveyi
to chitin.
Environmental Factors Affecting the Occurrence of Mycobacteria in Brook Waters
Authors: Iivanainen E. K. , Martikainen P. J. , Väänänen P. K. , Katila M.-L.
Published: January 2020

To evaluate the impact of environmental factors on the occurrence of environmental mycobacteria, viable counts of mycobacteria were measured in samples of brook water collected from 53 drainage areas located in a linear belt crossing Finland at 63° north latitude. The numbers of mycobacteria were correlated with characteristics of the drainage area, climatic parameters, chemical and physical characteristics of the water, and counts of other heterotrophic bacteria in the water. The numbers of mycobacteria in the water ranged from 10 to 2,200 CFU/liter. The counts correlated positively (
P
< 0.001) with the presence of peatlands, precipitation data, chemical oxygen demand, water color, and concentrations of Fe, Al, Cu, Co, and Cr. The mycobacterial counts correlated negatively (
P
< 0.001) with water pH, whereas other heterotrophic bacterial counts lacked any correlation with pH. A linear regression model with four independent variables (i.e., peatlands in the drainage area, chemical oxygen demand, concentration of potassium, and pH) explained 83% of the variation in mycobacterial counts in brook waters. Our results suggest that acidification may enhance the growth of environmental mycobacteria.
Effect of phenotypic plasticity on epiphytic survival and colonization by Pseudomonas syringae
Authors: Wilson M , Lindow S E
Published: January 2020

The bacterial epiphyte Pseudomonas syringae MF714R was cultured on agar or in broth or collected from colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants. Cells cultured in liquid medium survived the least well after inoculation of leaf surfaces under all conditions. Cells cultured in solid medium exhibited the highest percent survival and desiccation tolerance in the growth chamber but generally survived less well in the field than did cells harvested from plants. Cells harvested from plants and inoculated onto plants in the field usually exhibited the highest percent survival, started to increase in population earlier, and reached a higher number than did cells cultured in vitro. Differences in field survival were apparently not attributable to differential UV tolerance. The observed effects of phenotypic plasticity on epiphytic survival and colonization should be considered in risk assessment studies, in studies of bacterial epidemiology, and in the use of microbial antagonists for biological pest control.
Dissolved Nitrogen Uptake by a Cyanobacterial Bloom (
Anabaena flos-aquae
) in a Subarctic Lake
Authors: Gu Binhe , Alexander Vera
Published: January 2020

Uptake of dissolved nitrogen (NH
4
+
+ NO
3
-
+ urea + N
2
) by a cyanobacterial [
Anabaena flos-aquae
(Lyngb.)] De Brèb population in Smith Lake, Alaska, was measured every 2 to 4 days during the spring of 1990. Total dissolved nitrogen uptake ranged from 0.34 to 24.75 μmol liter
-1
h
-1
, with a mean of 5.75 μmol liter
-1
h
-1
; the euphotic zone accounted for 91% of the uptake. The mean turnover time for dissolved combined nitrogen (NH
4
+
+ NO
3
-
+ urea) in the euphotic zone was less than 14 h, and that for NH
4
+
was only 3.6 h. The mean relative preference indices for NH
4
+
(2.4), NO
3
-
(0.4), and urea (0.5) established NH
4
+
as the preferred nitrogenous nutrient. The uptake rates were apparently dependent on biomass, temperature, and light. Regeneration, probably due to zooplankton excretion and bacterial remineralization of dissolved organic nitrogen, was the main source of NH
4
+
for the cyanobacterial growth. The high half-saturation constant for NH
4
+
with low ambient NH
4
+
concentration nevertheless resulted in the simultaneous utilization of several forms of nitrogen.
Gene Expression per Gene Dose, a Specific Measure of Gene Expression in Aquatic Microorganisms
Authors: Pichard S. L. , Paul J. H.
Published: January 2020

A method for the measurement of specific levels of gene expression that combines target mRNA and target DNA quantitation has been developed. The use of target gene dose as a normalizing factor for mRNA provides an alternative to 16S or 23S rRNA, which are unsuitable for use in the environment because of their presence in nontarget organisms. Both target mRNA and DNA are recovered from replicate samples and detected by using antisense and sense single-stranded RNA gene probes. For efficient mRNA recovery, the use of Millipore Durapore filters and multiple extractions was necessary. Quantitation was performed by radiometric detection by using a β-scanner and comparison of the sample signal against target mRNA and DNA standard curves. This method enabled the measurement of expression of the catechol-2,3-dioxygenase gene (
xylE
) contained on the thermoregulated plasmid pLV1013 in a marine
Vibrio
strain in culture and in the environment. In studies of the relationship between mRNA levels and enzyme activities, the appearance of enzyme activity lagged behind
xylE
mRNA synthesis by an hour after temperature induction. This suggests that mRNA analysis is well suited for determining rapid regulation of microbial gene expression at the transcriptional level in water column microbial populations.
DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae
Authors: Barta T M , Kinscherf T G , Uchytil T F , Willis D K
Published: January 2020

The tblA gene of Pseudomonas syringae is required for tabtoxin biosynthesis and is under the control of a regulatory gene, lemA. We have determined the nucleotide sequence of the tblA gene and identified the 5' end of the tblA gene transcript. The sequence of the tblA gene was identified to that of the recently reported open reading frame 1 gene of the tabA region of the BR2 chromosome. The open reading frame of the tblA gene potentially encodes a protein of 231 amino acids. mRNA from the tblA gene was detected at all phases of cells grown in minimal medium. This result is correlated with the constitutive production of tabtoxinine-beta-lactam (the biologically active part of the toxin) by P. syringae BR2R in minimal medium, as quantitated by a phenylisothiocyanate derivatization method.
Fluorinated Vitamin B
12
Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a
19
F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions
Authors: Stupperich Erhard , Eisinger Hans-Jürgen , Kerssebaum Rainer , Nexø Ebba
Published: January 2020

The homoacetogenic bacterium
Sporomusa ovata
synthesized the vitamin B
12
analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly,
Propionibacterium freudenreichii
and
Methanobacterium thermoautotrophicum
synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B
12
. Hence, it is possible to use
19
F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.
Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520
Authors: Tsujibo H , Minoura K , Miyamoto K , Endo H , Moriwaki M , Inamori Y
Published: January 2020

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.
Dipicolinic Acid Greatly Enhances Production of Spore Photoproduct in Bacterial Spores upon UV Irradiation
Authors: Setlow Barbara , Setlow Peter
Published: January 2020

Formation of the spore photoproduct (SP) (5-thyminyl-5,6-dihydrothymine) in DNA of dormant spores of
Bacillus subtilis
upon UV irradiation is due to binding of α/β-type small, acid-soluble proteins (SASP). However, the yield of SP as a function of UV fluence is ∼15-fold higher in spores than in an α/β-type-SASP-DNA complex in vitro. The yield of SP as a function of UV fluence in forespore DNA from mutants which make α/β-type SASP but not dipicolinic acid (DPA) was 10 to 20 times lower than that in dormant spores. Furthermore, the yield of SP as a function of UV fluence in an α/β-type-SASP-DNA complex in vitro was increased sixfold by DPA. These data provide further support for the idea that the high DPA level in dormant spores increases the yield of SP as a function of UV fluence and thereby sensitizes spores to UV.
The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms
Authors: Liu W , Hansen J N
Published: January 2020

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.
Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences
Authors: Kane M D , Poulsen L K , Stahl D A
Published: January 2020

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.
Purification and Properties of an
S
-Adenosylmethionine: 2,4-Disubstituted Phenol
O
-Methyltransferase from
Phanerochaete chrysosporium
Authors: Coulter Catherine , Kennedy James T. , McRoberts W. Colin , Harper David B.
Published: January 2020

An enzyme catalyzing the
O
-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by
S
-adeno-sylmethionine was isolated from
Phanerochaete chrysosporium
and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55�C. The

K
m

values for acetovanillone and
S
-adenosylmethionine were 34 and 99 μM, respectively.
S
-Adenosylhomocysteine acted as a powerful competitive inhibitor of
S
-adenosylmethionine, with a

K
i

of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the
O
-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.
Dissimilatory Fe(III) Reduction by the Marine Microorganism
Desulfuromonas acetoxidans
Authors: Roden Eric E. , Lovley Derek R.
Published: January 2020

The ability of the marine microorganism
Desulfuromonas acetoxidans
to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer
Geobacter metallireducens.
Washed cell suspensions of the type strain of
D. acetoxidans
reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The
c
-type cytochrome(s) of
D. acetoxidans
was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate.
D. acetoxidans
grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe
3
O
4
) and siderite (FeCO
3
) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO
3
) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to
D. acetoxidans, G. metallireducens
could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S
0
reduction.
D. acetoxidans
is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus,
D. acetoxidans
provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.
Regulation and Characterization of Xylanolytic Enzymes of
Thermoanaerobacterium saccharolyticum
B6A-RI
Authors: Lee Yong-Eok , Lowe Sue E. , Zeikus J. Gregory
Published: January 2020

During growth on xylan and xylose
Thermoanaerobacterium saccharolyticum
B6A-RI produced endoxylanase, β-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predominantly cell associated; however, during growth on limiting concentrations of xylan, the majority of endoxylanase activity was extracellular rather than cell associated. Endoxylanase, β-xylosidase, and arabinofuranosidase activities were induced by xylan, xylose, and arabinose, respectively. Acetyl esterase activity was constitutive, and endoxylanase activity was catabolite repressed by glucose. Extracellular endoxylanase existed as a high-molecular-weight complex (molecular weight, more than 10
6
). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms, the crude endoxylanase complex was composed of at least six activity bands. Endoxylanase was purified by gel filtration with Sephacryl S-300 and affinity chromatography with xylan coupled to Sepharose CL-4B preequilibrated to 45�C with 50 mM sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single area of endoxylanase activity was identified on the zymogram; when this activity was analyzed by SDS-PAGE, it was composed of a major protein with a molecular weight of approximately 160,000 and a minor protein with a molecular weight of approximately 130,000. The endoxylanase activity stained with Schiff's reagent, indicative of glycoproteins, displayed a specific activity of 41 U/mg of protein on xylan, and had pH and temperature optima of 6.0 and 70�C, respectively.
Evaluation of colilert-marine water for detection of total coliforms and Escherichia coli in the marine environment
Authors: Palmer C J , Tsai Y L , Lang A L , Sangermano L R
Published: January 2020

A test that allows for early detection of fecally contaminated coastal water would enhance public health protection. Colilert-Marine Water (Colilert-MW; Environetics, Branford, Conn.) is a rapid 24-h test that has recently been developed to detect total coliforms and Escherichia coli in coastal water. We performed a premarketing evaluation of the Colilert-MW product, testing it in parallel with the multiple tube fermentation (MTF) method for 86 coastal water samples in southern California. Statistical analysis was performed by using paired t tests and linear regression. Bacterial isolates were evaluated by biochemical and genetic analysis. The results of this study showed a strong correlation between the traditional MTF and the Colilert-MW method for detection of total coliforms (r = 0.95) and E. coli (r = 0.89) in ocean water samples. Paired t-test results indicated that the Colilert-MW and MTF were equivalent in detecting E. coli and that the Colilert-MW may be more sensitive in the detection of total coliforms. We conclude that Colilert-MW would be a useful tool with which to monitor coastal beach water.