bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of an Operon Encoding Two
c
-Type Cytochromes, an

aa
3

-Type Cytochrome Oxidase, and Rusticyanin in
Thiobacillus ferrooxidans
ATCC 33020
Authors: Appia-Ayme Corinne , Guiliani Nicolas , Ratouchniak Jeanine , Bonnefoy Violaine
Published: December 2019

Despite the importance of
Thiobacillus ferrooxidans
in bioremediation and bioleaching, little is known about the genes encoding electron transfer proteins implicated in its energetic metabolism. This paper reports the sequences of the four
cox
genes encoding the subunits of an
aa
3
-type cytochrome
c
oxidase. These genes are in a locus containing four other genes:
cyc2
, which encodes a high-molecular-weight cytochrome
c
;
cyc1
, which encodes a
c
4
-type cytochrome (
c
552
); open reading frame 1, which encodes a putative periplasmic protein of unknown function; and
rus
, which encodes rusticyanin. The results of Northern and reverse transcription-PCR analyses indicated that these eight genes are cotranscribed. Two transcriptional start sites were identified for this operon. Upstream from each of the start sites was a ς70-type promoter recognized in
Escherichia coli
. While transcription in sulfur-grown
T. ferrooxidans
cells was detected from the two promoters, transcription in ferrous-iron-grown
T. ferrooxidans
cells was detected only from the downstream promoter. The cotranscription of seven genes encoding redox proteins suggests that all these proteins are involved in the same electron transfer chain; a model taking into account the biochemistry and the genetic data is discussed.
Transposon-Induced Mutations in Two Loci of
Listeria monocytogenes
Serotype 1/2a Result in Phage Resistance and Lack of
N
-Acetylglucosamine in the Teichoic Acid of the Cell Wall
Authors: Tran Huyen L. , Fiedler F. , Hodgson D. A. , Kathariou S.
Published: December 2019

Teichoic acid-associated
N
-acetylglucosamine and rhamnose have been shown to serve as phage receptors in
Listeria monocytogenes
serotype 1/2a. We generated and characterized two single-copy Tn
916
ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified
ptsHI
locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was
clpC
, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in
N
-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.
Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards
Authors: Schnabel Elise L. , Jones Alan L.
Published: December 2019

The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc
r
) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc
r
determinants. Tc
r
was determined to be due to the presence of Tet B in
Pantoea agglomerans
and other members of the family
Enterobacteriacae
and Tet A, Tet C, or Tet G in most
Pseudomonas
isolates. The cause of Tc
r
was not identified in 16% of the isolates studied. The Tc
r
genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn
5393
. Transposable elements with Tc
r
determinants were detected by entrapment following introduction into
Escherichia coli
. Tet B was found within Tn
10
. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn
10
; one had IS
911
located within IS
10
-R and one had Tn
1000
located upstream of Tet B. Tet A was found within a novel variant of Tn
1721
, named Tn
1720
, which lacks the left-end
orfI
of Tn
1721
. Tet C was located within a 19-kb transposon, Tn
1404
, with transposition genes similar to those of Tn
501
, streptomycin (
aadA2
) and sulfonamide (
sulI
) resistance genes within an integron, Tet C flanked by direct repeats of IS
26
, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.
PCR Amplification of the Gene
acmA
Differentiates
Lactococcus lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
Authors: Garde Sonia , Babin Mar , Gaya Pilar , Nuñez Manuel , Medina Margarita
Published: December 2019

The occurrence of the
acmA
gene, encoding the lactococcal
N
-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the
acmA
gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with
Lactococcus lactis
subspecies.
L. lactis
subsp.
lactis
exhibits both the expected 1,131-bp product and the additional amplicon, whereas
L. lactis
subsp.
cremoris
exhibits a single 1,131-bp fragment.
Influence of Different Electron Donors and Acceptors on Dehalorespiration of Tetrachloroethene by
Desulfitobacterium frappieri
TCE1
Authors: Gerritse Jan , Drzyzga Oliver , Kloetstra Geert , Keijmel Mischa , Wiersum Luit P. , Hutson Roger , Collins Matthew D. , Gottschal Jan C.
Published: December 2019

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 μm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35°C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of
Desulfitobacterium frappieri
, because it exhibited 99.7% relatedness to the
D. frappieri
type strain, strain PCP-1. Growth with H
2
, formate,
l
-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H
2
) are oxidized to acetate and CO
2
. When
l
-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce
cis
-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g.,
Desulfitobacterium
sp. strain PCE1 and
Desulfitobacterium hafniense
) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 μmol of chloride released · min
−1
· mg of protein
−1
). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.
Dynamic Interaction of
Trichoderma reesei
Cellobiohydrolases Cel6A and Cel7A and Cellulose at Equilibrium and during Hydrolysis
Authors: Palonen Hetti , Tenkanen Maija , Linder Markus
Published: December 2019

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two
Trichoderma reesei
cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.
cis
-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from
Pseudomonas
sp. Strain P51, Expressed in
Escherichia coli
DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions
Authors: Raschke Henning , Fleischmann Thomas , Van Der Meer Jan Roelof , Kohler Hans-Peter E.
Published: December 2019

cis
-Chlorobenzene dihydrodiol dehydrogenase (CDD) from
Pseudomonas
sp. strain P51, cloned into
Escherichia coli
DH5α(pTCB149) was able to oxidize
cis
-dihydrodihydroxy derivatives (
cis
-dihydrodiols) of dihydronaphthalene, indene, and four
para
-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of
cis
-1,2-indandiol, only the (+)-
cis
-(1
R
,2
S
) enantiomer was oxidized; the (−)-
cis
-(
S
,2
R
) enantiomer remained unchanged. CDD oxidized both enantiomers of
cis
-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-
cis
-(1
S
,2
R
) enantiomer was delayed until the (−)-
cis
-(1
R
,2
S
) enantiomer was completely depleted. When incubated with nonracemic mixtures of
para
-substituted
cis
-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1
S
) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-
cis
-(1
R
,2
S
)-dihydroxy-1,2-dihydronaphthalene, (+)-
cis
-monochlorobiphenyl dihydrodiols, and (+)-
cis
-toluene dihydrodiol to the corresponding catechols.
A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon
Pyrococcus kodakaraensis
KOD1
Authors: Tanaka Takeshi , Fujiwara Shinsuke , Nishikori Shingo , Fukui Toshiaki , Takagi Masahiro , Imanaka Tadayuki
Published: December 2019

We have found that the hyperthermophilic archaeon
Pyrococcus kodakaraensis
KOD1 produces an extracellular chitinase. The gene encoding the chitinase (
chiA
) was cloned and sequenced. The
chiA
gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from
Bacillus circulans
WL-12 and chitinase from
Streptomyces erythraeus
(ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type.
chiA
was expressed in
Escherichia coli
cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.
Mitochondrial Function in Cell Wall Glycoprotein Synthesis in
Saccharomyces cerevisiae
NCYC 625 (Wild Type) and [
rho
0
] Mutants
Authors: Iung Annie Rakotoarivony , Coulon Joël , Kiss Ferenc , Ekome Jacques Ngondi , Vallner Judit , Bonaly Roger
Published: December 2019

We studied phosphopeptidomannans (PPMs) of two
Saccharomyces cerevisiae
NCYC 625 strains (
S. diastaticus
): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [
rho
0
] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [
rho
0
] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [
rho
0
] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.
Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from
Xanthophyllomyces dendrorhous
Authors: Visser Hans , de Bont Jan A. M. , Verdoes Jan C.
Published: December 2019

The epoxide hydrolase (EH)-encoding gene (
EPH1
) from the basidiomycetous yeast
Xanthophyllomyces dendrorhous
was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The
EPH1
gene was not essential for growth of
X. dendrorhous
in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in
Escherichia coli
. A sixfold increase in specific activity was observed when we used resting cells rather than
X. dendrorhous
. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the
X. dendrorhous
EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.
Novel Interaction between Laccase and Cellobiose Dehydrogenase during Pigment Synthesis in the White Rot Fungus
Pycnoporus cinnabarinus
Authors: Temp Ulrike , Eggert Claudia
Published: December 2019

When glucose is the carbon source, the white rot fungus
Pycnoporus cinnabarinus
produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When
P. cinnabarinus
was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of
P. cinnabarinus
; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome
c
. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from
P. cinnabarinus
. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.
Composition and Enzymatic Activity of the Extracellular Matrix Secreted by Germlings ofBotrytis cinerea
Authors: Doss Robert P.
Published: December 2019

Germlings ofBotrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260–265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) andmyo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis ofp-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.
Green Fluorescent Protein as a Noninvasive Stress Probe in Resting
Escherichia coli
Cells
Authors: Cha Hyung Joon , Srivastava Ranjan , Vakharia Vikram N. , Rao Govind , Bentley William E.
Published: December 2019

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate
Escherichia coli
cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor ς
32
, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene

gfp
uv

. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-β-
d
-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the
E. coli
cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.
A Strategy for Detection of Viruses in Groundwater by PCR
Authors: Abbaszadegan Morteza , Stewart Peter , LeChevallier Mark
Published: December 2019

We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry’s need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 μl) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a “snapshot” of the quality of the groundwater being sampled, PCR seems to be a desirable rapid initial screening tool.
Multiple α-Glucoside Transporter Genes in Brewer’s Yeast
Authors: Jespersen Lene , Cesar Lene B. , Meaden Philip G. , Jakobsen Mogens
Published: December 2019

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous
MAL
loci (
MAL1
to
MAL4
and
MAL6
). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain
MAL11
and
MAL31
sequences, and only one of these strains lacked
MAL41. MAL21
was not present in the five ale strains and 12 of the lager strains.
MAL61
was not found in any of the yeast strains. In three of the lager strains, there was evidence that
MAL
transporter gene sequences occurred on chromosomes other than those known to carry
MAL
loci. Sequences corresponding to the
AGT1
gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of
AGT1
were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI.
AGT1
appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.
Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from
Pseudomonas fluorescens
with High Activity towards Lactones
Authors: Khalameyzer V. , Fischer I. , Bornscheuer U. T. , Altenbuchner J.
Published: December 2019

A genomic library of
Pseudomonas fluorescens
DSM 50106 in a λRESIII phage vector was screened in
Escherichia coli
K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of
P. fluorescens
DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in
E. coli
. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to ɛ-caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.
Manganese Deficiency Can Replace High Oxygen Levels Needed for Lignin Peroxidase Formation by
Phanerochaete chrysosporium
Authors: Rothschild Nathan , Levkowitz Ayala , Hadar Yitzhak , Dosoretz Carlos G.
Published: December 2019

The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in
Phanerochaete chrysosporium
were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.
Effect of Acetate on Molecular and Physiological Aspects of
Clostridium beijerinckii
NCIMB 8052 Solvent Production and Strain Degeneration
Authors: Chen Chih-Kuang , Blaschek Hans P.
Published: December 2019

The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by
Clostridium beijerinckii
NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in
C. beijerinckii
NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a
sol
operon and confirmed the presence of a
ptb-buk
operon in
C. beijerinckii
NCIMB 8052. In addition to CoA transferase,
C. beijerinckii
NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated
C. beijerinckii
NCIMB 8052 indicated that
C. beijerinckii
NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the
sol
operon, which ensures solvent production and prevents strain degeneration in
C. beijerinckii
NCIMB 8052.
Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA
Authors: Acinas Silvia G. , Antón Josefa , Rodríguez-Valera Francisco
Published: December 2019

In a previous study (S. G. Acinas, F. Rodrı́guez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria,
Alteromonas macleodii
, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γ
Proteobacteria
predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α
Proteobacteria
of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the
A. macleodii
cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.
Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype
Authors: Pag Ulrike , Heidrich Christoph , Bierbaum Gabriele , Sahl Hans-Georg
Published: December 2019

The lantibiotic Pep5 is produced by
Staphylococcus epidermidis
5. Within its biosynthetic gene cluster, the immunity gene
pepI
, providing producer self-protection, is localized upstream of the structural gene
pepA
. Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876–2883, 1994). To study this phenomenon, we analyzed
pepA
and
pepI
transcription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of a
pepA
-expressing strain with
pepI
in
trans
did not result in phenotypic immunity or production of PepI. On the other hand, neither
pepA
nor its product was found to be involved in immunity, since suppression of the translation of the
pepA
mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of
pepI
from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability of
pepI
transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of −56.9 kJ/mol, localized downstream of
pepA
. We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.
Identification of Lactic Acid Bacteria from Chili Bo, a Malaysian Food Ingredient
Authors: Leisner Jørgen J. , Pot Bruno , Christensen Henrik , Rusul Gulam , Olsen John E. , Wee Bee Wah , Muhamad Kharidah , Ghazali Hasanah M.
Published: December 2019

Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28°C with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg
−1
(product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as
Lactobacillus plantarum
,
Lactobacillus fermentum
,
Lactobacillus farciminis
,
Pediococcus acidilactici
,
Enterococcus faecalis
, and
Weissella confusa
. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the
Lactobacillus casei/Pediococcus
group. Two strains were related to
Weissella
at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the
L. casei/Pediococcus
group and the
Weissella
group and species of
Carnobacterium
. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against
L. plantarum
ATCC 14917 and LMG 17682.
Genetic and Morphological Characterization of
Cladobotryum
Species Causing Cobweb Disease of Mushrooms
Authors: McKay Gareth J. , Egan Damian , Morris Elizabeth , Scott Carol , Brown Averil E.
Published: December 2019

Cladobotryum dendroides
(=
Dactylium dendroides
) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom,
Agaricus bisporus
. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four
Cladobotryum/Hypomyces
species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now
C. mycophilum
. This cobweb pathogen varies somewhat in conidial septation from published descriptions of
C. mycophilum
and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.
Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium
Shewanella
Strain Ac10: Gene Cloning and Enzyme Purification and Characterization
Authors: Kulakova Ljudmila , Galkin Andrey , Kurihara Tatsuo , Yoshimura Tohru , Esaki Nobuyoshi
Published: December 2019

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium
Shewanella
strain Ac10 was cloned in
Escherichia coli
. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of
E. coli
recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15°C. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.
Effect of Compressed Carbon Dioxide on Microbial Cell Viability
Authors: Debs-Louka E. , Louka N. , Abraham G. , Chabot V. , Allaf K.
Published: December 2019

In order to study the influence of compressed carbon dioxide, over a range of pressures (1.5 to 5.5 MPa) and exposure times (up to 7 h), on the survival of
Escherichia coli
,
Saccharomyces cerevisiae
, and
Enterococcus faecalis
, a new pressurizable reactor system was conceived. Microbial cells were inoculated onto a solid hydrophilic medium and treated at room temperature; their sensitivities to inactivation varied greatly. The CO
2
treatment had an enhanced efficiency in cell destruction when the pressure and the duration of exposure were increased. The effects of these parameters on the loss of viability was also studied by response-surface methodology. This study showed that a linear correlation exists between microbial inactivation and CO
2
pressure and exposure time, and in it models were proposed which were adequate to predict the experimental values. The end point acidity was measured for all the samples in order to understand the mechanism of microbial inactivation. The pHs of the treated samples did not vary, regardless of the experimental conditions. Other parameters, such as water content and pressure release time, were also investigated.
De Novo Synthesis of 4,5-Dimethoxycatechol and 2,5-Dimethoxyhydroquinone by the Brown Rot Fungus
Gloeophyllum trabeum
Authors: Paszczynski Andrzej , Crawford Ronald , Funk David , Goodell Barry
Published: December 2019

The new dimethoxycatechol 4,5-dimethoxy-1,2-benzenediol (DMC) and the new dimethoxyhydroquinone 2,5-dimethoxy-1,4-benzenediol (DMH) were isolated from stationary cultures of the brown rot fungus
Gloeophyllum trabeum
growing on a glucose mineral medium protected from light. The structure was elucidated by gas chromatography-mass spectrometry through comparison to a synthetic standard. Further confirmation was obtained by forming a dimethoxyoxazole derivative by condensation of DMC with methylene chloride and through examination of methylated derivatives. DMC and DMH may serve as ferric chelators, oxygen-reducing agents, and redox-cycling molecules, which would include functioning as electron transport carriers to Fenton’s reactions. Thus, they appear to be important components of the brown rot decay system of the fungus.
Changes in Bacterial and Eukaryotic Community Structure after Mass Lysis of Filamentous Cyanobacteria Associated with Viruses
Authors: van Hannen Erik J. , Zwart Gabriel , van Agterveld Miranda P. , Gons Herman J. , Ebert Jeannine , Laanbroek Hendrikus J.
Published: December 2019

During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the
Cytophagales
and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities.
Comparative Study of Biological Properties and Electrophoretic Characteristics of Lipopolysaccharide from Eel-Virulent and Eel-A virulent
Vibrio vulnificus
Strains
Authors: Biosca Elena G. , Collado Rosa M. , Oliver James D. , Amaro Carmen
Published: December 2019

In
Vibrio vulnificus
, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.
Decolorization and Detoxification of Extraction-Stage Effluent from Chlorine Bleaching of Kraft Pulp by
Rhizopus oryzae
Authors: Nagarathnamma R. , Bajpai Pratima
Published: December 2019

Rhizopus oryzae
, a zygomycete, was found to decolorize, dechlorinate, and detoxify bleach plant effluent at lower cosubstrate concentrations than the basidiomycetes previously investigated. With glucose at 1 g/liter, this fungus removed 92 to 95% of the color, 50% of the chemical oxygen demand, 72% of the adsorbable organic halide, and 37% of the extractable organic halide in 24 h at temperatures of 25 to 45°C and a pH of 3 to 5. Even without added cosubstrate the fungus removed up to 78% of the color. Monomeric chlorinated aromatic compounds were removed almost completely, and toxicity to zebra fish was eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The residue after treatment was not further improved by exposure to fresh
R. oryzae
mycelium.
Arbitrarily Primed PCR To Type
Vibrio
spp. Pathogenic for Shrimp
Authors: Goarant Cyrille , Merien Fabrice , Berthe Franck , Mermoud Isabelle , Perolat Philippe
Published: December 2019

A molecular typing study on
Vibrio
strains implicated in shrimp disease outbreaks in New Caledonia and Japan was conducted by using AP-PCR (arbitrarily primed PCR). It allowed rapid identification of isolates at the genospecies level and studies of infraspecific population structures of epidemiological interest. Clusters identified within the species
Vibrio penaeicida
were related to their area of origin, allowing discrimination between Japanese and New Caledonian isolates, as well as between those from two different bays in New Caledonia separated by only 50 km. Other subclusters of New Caledonian
V. penaeicida
isolates could be identified, but it was not possible to link those differences to accurate epidemiological features. This contribution of AP-PCR to the study of vibriosis in penaeid shrimps demonstrates its high discriminating power and the relevance of the epidemiological information provided. This approach would contribute to better knowledge of the ecology of
Vibrio
spp. and their implication in shrimp disease in aquaculture.
Heat Resistance of Native and Demineralized Spores of
Bacillus subtilis
Sporulated at Different Temperatures
Authors: Palop Alfredo , Sala Francisco J. , Condón Santiago
Published: December 2019

Demineralization reduced heat resistance of
B. subtilis
spores, but the pattern and magnitude of the reduction depended on sporulation temperature and on heating menstruum pH. The differences in heat resistance of native spores caused by sporulation temperature almost disappeared after demineralization. Demineralized spores were still susceptible to the heat-sensitizing effect of acidic pH.
Contrasting Effects of a Nonionic Surfactant on the Biotransformation of Polycyclic Aromatic Hydrocarbons to
cis
-Dihydrodiols by Soil Bacteria
Authors: Allen Christopher C. R. , Boyd Derek R. , Hempenstall Francis , Larkin Michael J. , Sharma Narain D.
Published: December 2019

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria,
Pseudomonas
sp. strain 9816/11 and
Sphingomonas yanoikuyae
B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate
cis
-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain
S. yanoikuyae
B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.