bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of Poly(3-Hydroxybutyric Acid-Co-4-Hydroxybutyric Acid) and Poly(4-Hydroxybutyric Acid) without Subsequent Degradation by
Hydrogenophaga pseudoflava
Authors: Choi Mun Hwan , Yoon Sung Chul , Lenz Robert W.
Published: December 2019

A
Hydrogenophaga pseudoflava
strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from γ-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution
13
C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on γ-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on γ-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from γ-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to γ-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.
Phylogenetic Analysis of
Cryptosporidium
Parasites Based on the Small-Subunit rRNA Gene Locus
Authors: Xiao Lihua , Escalante Lillian , Yang Chunfu , Sulaiman Irshad , Escalante Anannias A. , Montali Richard J. , Fayer Ronald , Lal Altaf A.
Published: December 2019

Biological data support the hypothesis that there are multiple species in the genus
Cryptosporidium
, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of
Cryptosporidium parvum
,
Cryptosporidium baileyi
,
Cryptosporidium muris
, and
Cryptosporidium serpentis
and performed a phylogenetic analysis of the genus
Cryptosporidium
. Our study revealed that the genus
Cryptosporidium
contains the phylogenetically distinct species
C. parvum
,
C. muris
,
C. baileyi
, and
C. serpentis
, which is consistent with the biological characteristics and host specificity data. The
Cryptosporidium
species formed two clades, with
C. parvum
and
C. baileyi
belonging to one clade and
C. muris
and
C. serpentis
belonging to the other clade. Within
C. parvum
, human genotype isolates and guinea pig isolates (known as
Cryptosporidium wrairi
) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A
C. muris
isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between
C. serpentis
isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.
The Alkene Monooxygenase from
Xanthobacter
Strain Py2 Is Closely Related to Aromatic Monooxygenases and Catalyzes Aromatic Monohydroxylation of Benzene, Toluene, and Phenol
Authors: Zhou Ning-Yi , Jenkins Alister , Chan Kwo Chion Chan K. N. , Leak David J.
Published: December 2019

The genes encoding the six polypeptide components of the alkene monooxygenase from
Xanthobacter
strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are
aamA
, encoding the 497-residue oxygenase α-subunit (XamoA);
aamB
, encoding the 88-residue oxygenase γ-subunit (XamoB);
aamC
, encoding the 122-residue ferredoxin (XamoC);
aamD
, encoding the 101-residue coupling or effector protein (XamoD);
aamE
, encoding the 341-residue oxygenase β-subunit (XamoE); and
aamF
, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of ς
54
(RpoN)-dependent promoters was identified upstream of the
aamA
gene. Detailed comparison of XamoA with the oxygenase α-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from
Rhodococcus rhodochrous
B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases,
Xanthobacter
strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of
o
-,
m
-, and
p
-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies
Xanthobacter
strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the
aam
gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.
Ferrioxamine-Mediated Iron(III) Utilization bySalmonella enterica
Authors: Kingsley Robert A. , Reissbrodt Rolf , Rabsch Wolfgang , Ketley Julian M. , Tsolis Renée M. , Everest Paul , Dougan Gordon , Bäumler Andreas J. , Roberts Mark , Williams Peter H.
Published: December 2019

Utilization of ferrioxamines as sole sources of iron distinguishesSalmonella entericaserotypes Typhimurium and Enteritidis from a number of related species, includingEscherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G byS. entericaserotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressiblefoxAgene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of atonBstrain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of thefoxAgene in the genusSalmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) andSalmonella bongori(formerly subspecies V).S. entericaserotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in thefoxAgene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolarfoxAmutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to thefoxA+parent. In addition, afoxAmutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with thefoxAmutant were protected against subsequent challenge by thefoxA+parent strain.
High-Rate Anaerobic Treatment of Wastewater at Low Temperatures
Authors: Lettinga Gatze , Rebac Salih , Parshina Sofia , Nozhevnikova Alla , van Lier Jules B. , Stams Alfons J. M.
Published: December 2019

Anaerobic treatment of a volatile fatty acid (VFA) mixture was investigated under psychrophilic (3 to 8°C) conditions in two laboratory-scale expanded granular sludge bed reactor stages in series. The reactor system was seeded with mesophilic methanogenic granular sludge and fed with a mixture of VFAs. Good removal of fatty acids was achieved in the two-stage system. Relative high levels of propionate were present in the effluent of the first stage, but propionate was efficiently removed in the second stage, where a low hydrogen partial pressure and a low acetate concentration were advantageous for propionate oxidation. The specific VFA-degrading activities of the sludge in each of the modules doubled during system operation for 150 days, indicating a good enrichment of methanogens and proton-reducing acetogenic bacteria at such low temperatures. The specific degradation rates of butyrate, propionate, and the VFA mixture amounted to 0.139, 0.110, and 0.214 g of chemical oxygen demand g of volatile suspended solids−1day−1, respectively. The biomass which was obtained after 1.5 years still had a temperature optimum of between 30 and 40°C.
Real-Time Monitoring of
Escherichia coli
O157:H7 Adherence to Beef Carcass Surface Tissues with a Bioluminescent Reporter
Authors: Siragusa Gregory R. , Nawotka Kevin , Spilman Stanley D. , Contag Pamela R. , Contag Christopher H.
Published: December 2019

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of
Escherichia coli
O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (
lux
) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete
lux
operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (
r
2
= 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.
Genomic Relationships between
Enterococcus faecium
Strains from Different Sources and with Different Antibiotic Resistance Profiles Evaluated by Restriction Endonuclease Analysis of Total Chromosomal DNA Using
Eco
RI and
Pvu
II
Authors: Quednau M. , Ahrné S. , Molin G.
Published: December 2019

Forty-seven
Enterococcus faecium
strains from different sources were evaluated by restriction endonuclease analysis (REA) of total chromosomal DNA. Strains from chicken, pork, and humans were clearly divided into separate clusters, whereas strains from different countries, strains with different antibiotic resistance profiles, or clinical and healthy-subject strains were not.
Different Strategies for Molecular Differentiation of
Mycobacterium bovis
Strains Isolated in Sardinia, Italy
Authors: Sechi Leonardo A. , Leori Guido , Lollai Stefano A. , Duprè Ilaria , Molicotti Paola , Fadda Giovanni , Zanetti Stefania
Published: December 2019

Different genetic markers were used to analyze 22
Mycobacterium bovis
strains isolated from cattle in Sardinia and one human isolate. IS
6110
DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with
Hae
III and
Pvu
II produced five and seven patterns, respectively. PCR with the (GTG)
5
oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.
Enhanced Degradation of Polyvinyl Alcohol by
Pycnoporus cinnabarinus
after Pretreatment with Fenton’s Reagent
Authors: Larking Daniel M. , Crawford Russell J. , Christie Gregor B. Y. , Lonergan Greg T.
Published: December 2019

Degradation of polyvinyl alcohol (PVA) was investigated by using a combination of chemical treatment with Fenton’s reagent and biological degradation with the white rot fungus
Pycnoporus cinnabarinus
. Inclusion of the chemical pretreatment resulted in greater degradation of PVA than the degradation observed when biological degradation alone was used.
Activation and Inactivation of
Pseudomonas stutzeri
Methylbenzene Catabolism Pathways Mediated by a Transposable Element
Authors: Bolognese Fabrizio , di Lecce Cinzia , Galli Enrica , Barbieri Paola
Published: December 2019

The arrangement of the genes involved in
o
-xylene,
m
-xylene, and
p
-xylene catabolism was investigated in three
Pseudomonas stutzeri
strains: the wild-type strain OX1, which is able to grow on
o
-xylene but not on the
meta
and
para
isomers; the mutant M1, which grows on
m
-xylene and
p
-xylene but is unable to utilize the
ortho
isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed IS
Ps1
, which inactivates the
m
-xylene and
p
-xylene catabolic pathway in
P. stutzeri
OX1 and the
o
-xylene catabolic genes in
P. stutzeri
M1, was detected. No IS was detected in the corresponding catabolic regions of the
P. stutzeri
R1 genome. IS
Ps1
is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family.
Feed Deprivation Affects Crop Environment and Modulates
Salmonella enteritidis
Colonization and Invasion of Leghorn Hens
Authors: Durant Juliet A. , Corrier Donald E. , Byrd J. Allen , Stanker Larry H. , Ricke Steven C.
Published: December 2019

Leghorn hens over 50 weeks of age were assigned to two treatment groups designated as either unmolted controls or molted. A forced molt was induced by a 9-day feed withdrawal, and each hen was challenged orally with 10
5
Salmonella enteritidis
organisms on day 4 of feed withdrawal. On days 4 and 9 of molt, the numbers of lactobacilli and the concentrations of lactate, acetate, propionate, and butyrate, and total volatile fatty acids in the crops decreased while crop pH increased significantly (
P
< 0.05) in the molted hens compared to the controls.
S. enteritidis
crop and cecal colonization, in addition to spleen and liver invasion, increased significantly (
P
< 0.05) in the molted hens compared to the controls. The invasive phenotype of
Salmonella
spp. is complex and requires several virulence genes which are regulated by the transcriptional activator HilA. Samples of the crop contents from the molted and unmolted birds were pooled separately, centrifuged, and filter sterilized. The sterile crop contents were then used to measure the expression of
hilA
. By using a
lacZY
transcriptional fusion to the
hilA
gene in
S. enteritidis
, we found that
hilA
expression was 1.6- to 2.1-fold higher in the crop contents from molted birds than in those from control birds in vitro. The results of the study suggest that the changes in the microenvironment of the crop caused by feed deprivation are important regulators of
S. enteritidis
survival and influence the susceptibility of molted hens to
S. enteritidis
infections. Furthermore, our in vitro results on the expression of
hilA
suggest that the change in crop environment during feed withdrawal has the potential to significantly affect virulence by increasing the expression of genes necessary for intestinal invasion.
Characterization of Six Bacteriophages ofSerratia liquefaciensCP6 Isolated from the Sugar Beet Phytosphere
Authors: Ashelford Kevin E. , Fry John C. , Bailey Mark J. , Jeffries Aaron R. , Day Martin J.
Published: December 2019

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgarisvar. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciensCP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the familySiphoviridae, while ΦCP6-4 exhibited the morphology of the familyPodoviridae. The other phages were members of the familyMyoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10−9to 3.9 × 10−7, whereas ΦCP6-4 could not transduceS. liquefaciensCP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.
Cell-Wall-Bound Proteinase of
Lactobacillus delbrueckii
subsp.
lactis
ACA-DC 178: Characterization and Specificity for β-Casein
Authors: Tsakalidou E. , Anastasiou R. , Vandenberghe I. , van Beeumen J. , Kalantzopoulos G.
Published: December 2019

Lactobacillus delbrueckii
subsp.
lactis
ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca
2+
-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal P
I
-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from
L. delbrueckii
subsp.
lactis
ACA-DC 178 liberates four main peptides from β-casein, which have been identified.
Antagonism between Cry1Ac1 and Cyt1A1 Toxins of
Bacillus thuringiensis
Authors: del Rincón-Castro M. Cristina , Barajas-Huerta José , Ibarra Jorge E.
Published: December 2019

Most strains of the insecticidal bacterium
Bacillus thuringiensis
have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in
B. thuringiensis
subsp.
israelensis
. In this paper we describe a novel joint activity of toxins from different strains of
B. thuringiensis
. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from
B. thuringiensis
subsp.
kurstaki
, Cyt1A1 from
B. thuringiensis
subsp.
israelensis
, and
Trichoplusia ni
BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC
50
s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC
50
s were obtained. All of these LC
50
s were significantly higher than the expected LC
50
s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC
50
of Cry1Ac1 was 2.46 ng/cm
2
of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC
50
s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm
2
of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm
2
of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between
B. thuringiensis
toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of
B. thuringiensis
(
B. thuringiensis
subsp.
kurstaki
and
B. thuringiensis
subsp.
israelensis
) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.
Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family
Rhizobiaceae
and Their Roles in Osmoprotection
Authors: Boncompagni Eric , Østerås Magne , Poggi Marie-Christine , le Rudulier Daniel
Published: December 2019

The role of glycine betaine and choline in osmoprotection of various
Rhizobium
,
Sinorhizobium
,
Mesorhizobium
,
Agrobacterium
, and
Bradyrhizobium
reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of
Rhizobium tropici
,
Sinorhizobium meliloti
,
Sinorhizobium fredii
,
Rhizobium galegae
,
Agrobacterium tumefaciens
,
Mesorhizobium loti
, and
Mesorhizobium huakuii
, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as
Rhizobium leguminosarum
(all biovars),
Agrobacterium rhizogenes
,
Rhizobium etli
, and
Bradyrhizobium japonicum
. Except for
B. japonicum
, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in
R. leguminosarum
and
S. meliloti
or, more surprisingly, reduced in
R. tropici
,
S. fredii
, and
M. loti
. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of
B. japonicum
, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of
M. huakuii
and
B. japonicum
. The main physiological role of glycine betaine in the family
Rhizobiaceae
seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.
Rhodococcus erythropolis
DCL14 Contains a Novel Degradation Pathway for Limonene
Authors: van der Werf Mariët J. , Swarts Henk J. , de Bont Jan A. M.
Published: December 2019

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of
Rhodococcus erythropolis
by chemotaxonomic and genetic studies.
R. erythropolis
DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of
R. erythropolis
DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1
S
,2
S
,4
R
)-limonene-1,2-diol, (1
S
,4
R
)-1-hydroxy-2-oxolimonene, and (3
R
)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4
R
)-limonene degradation pathway. The opposite enantiomers [(1
R
,2
R
,4
S
)-limonene-1,2-diol, (1
R
,4
S
)-1-hydroxy-2-oxolimonene, and (3
S
)-3-isopropenyl-6-oxoheptanoate] were found in the (4
S
)-limonene degradation pathway, while accumulation of (1
R
,2
S
,4
S
)-limonene-1,2-diol from (4
S
)-limonene was also observed. These results show that
R. erythropolis
DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.
Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe
Butyrivibrio fibrisolvens
OR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A
Authors: Kalmokoff M. L. , Lu D. , Whitford M. F. , Teather R. M.
Published: December 2019

The ruminal anaerobe
Butyrivibrio fibrisolvens
OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (
bvi79A
) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of
bvi79A
was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of
bvi79A
or with degenerate oligonucleotides based on the predicted sequence of the second peptide.
Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by
Enterococcus faecium
BFE 900
Authors: Franz Charles M. A. P. , Worobo Randy W. , Quadri Luis E. N. , Schillinger Ulrich , Holzapfel Wilhelm H. , Vederas John C. , Stiles Michael E.
Published: December 2019

A purified bacteriocin produced by
Enterococcus faecium
BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by
E. faecium
T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb
Hin
dIII fragment and a 12.0-kb
Eco
RI chromosomal fragment. The genetic characteristics and production of EntB by
E. faecium
BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of
Carnobacterium piscicola
LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in
Enterococcus faecalis
ATCC 19433.
Analysis of 16S-23S rRNA Intergenic Spacer Regions of
Vibrio cholerae
and
Vibrio mimicus
Authors: Chun Jongsik , Huq Anwarul , Colwell Rita R.
Published: December 2019

Vibrio cholerae
identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related
Vibrio mimicus
. The two species share many genes coding for proteins, such as
ctxAB
, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of
V. cholerae
and
V. mimicus
. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNA
Glu
, tRNA
Lys
, and tRNA
Val
, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNA
Ile
and tDNA
Ala
, whereas the 500-bp fragment had single tDNA coding either tRNA
Glu
or tRNA
Ala
. Little variation, i.e., 0 to 0.4%, was found among
V. cholerae
O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and
V. mimicus
(2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of
V. cholerae
strains from
V. mimicus
. The PCR system developed was subsequently evaluated by using representatives of
V. cholerae
from environmental and clinical sources, and of other taxa, including
V. mimicus
. This study provides the first molecular tool for identifying the species
V. cholerae
.
Biodegradation of Cyclohexylamine by
Brevibacterium oxydans
IH-35A
Authors: Iwaki Hiroaki , Shimizu Masatake , Tokuyama Tai , Hasegawa Yoshie
Published: December 2019

A bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques. The strain isolated, strain IH-35A, was classified as a member of the genus
Brevibacterium
. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.
A Sandwiched-Culture Technique for Evaluation of Heterologous Protein Production in a Filamentous Fungus
Authors: Ásgeirsdóttir Sigrídur A. , Scholtmeijer Karin , Wessels Joseph G. H.
Published: December 2019

Aspergillus niger
is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of
Agaricus bisporus
in
A. niger
, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein.
Examination of Physiological and Molecular Factors Involved in Enhanced Solvent Production by
Clostridium beijerinckii
BA101
Authors: Chen Chih-Kuang , Blaschek Hans P.
Published: December 2019

The specific activities and the mRNA expression levels associated with coenzyme A transferase, acetoacetate decarboxylase, and butyraldehyde dehydrogenase were elevated in hyper-solvent-producing
Clostridium beijerinckii
BA101 during the exponential growth phase. The increase in expression of the
sol
operon and associated enzyme activities may be responsible for enhanced solvent production by
C. beijerinckii
BA101.
The Pediocin AcH Precursor Is Biologically Active
Authors: Ray Bibek , Schamber Robin , Miller Kurt W.
Published: December 2019

The properties of the pediocin AcH precursor, prepediocin AcH, have been studied to gain insight into how producer cells may protect themselves from the activity of intracellular prebacteriocins. The native 62-amino-acid precursor and the 44-amino-acid mature species were expressed in
Escherichia coli
host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the growth of the test bacterium
Listeria innocua
Lin11, indicating that the native precursor is biologically active. The two species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was ∼80% as active as the chimeric mature species. Of relevance to cell protection and pediocin AcH production, it was determined that the precursor is strongly susceptible to inactivation by reducing agents and to degradation by chymotrypsin and endogenous
E. coli
proteases. Taken together, the results indicate that the activity of prepediocin AcH may have to be controlled prior to secretion to prevent toxicity to the host. Perhaps producer cells avoid membrane damage by maintaining the precursor in a reduced inactive state or by degrading molecules whose secretion is delayed.
Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H
+
-ATPase Levels in Chemostat Cultures of
Lactococcus lactis
Authors: O’Sullivan Eilís , Condon Séamus
Published: December 2019

The acid tolerance response (ATR) of chemostat cultures of
Lactococcus lactis
subsp.
cremoris
NCDO 712 was dependent on the dilution rate and on the extracellular pH (pH
o
). A decrease in either the dilution rate or the pH
o
led to a decrease in the cytoplasmic pH (pH
i
) of the cells, and similar levels of acid tolerance were observed at any specific pH
i
irrespective of whether the pH
i
resulted from manipulation of the growth rate, manipulation of the pH
o
, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pH
o
of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pH
o
of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pH
i
. The rate of ATP formation was not correlated with pH
i
, but a good correlation between the cellular level of H
+
-ATPase and pH
i
was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pH
i
was established. Each pH
i
below 6.6 was characterized by unique levels of ATR, H
+
-ATPase, and ATP. High levels of H
+
-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H
+
-ATPase is one of the ATR proteins induced by acid pH
i
through growth at an acid pH
o
or a slow growth rate.
Two-Component Transcriptional Regulation of N -Acyl-Homoserine Lactone Production in
Pseudomonas aureofaciens
Authors: Chancey S. T. , Wood D. W. , Pierson L. S.
Published: December 2019

Production of phenazine antibiotics by the biological control bacterium
Pseudomonas aureofaciens
30-84 is regulated in part by the PhzI/PhzR
N
-acyl-homoserine lactone (AHL) response system (L. S. Pierson III, V. D. Keppenne, and D. W. Wood, J. Bacteriol. 176:3966–3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49–53, 1996). Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal
N
-hexanoyl-homoserine lactone. These mutants were not complemented by
phzI
,
phzR
, or the phenazine biosynthetic genes (
phzFABCD
) (L. S. Pierson III, T. Gaffney, S. Lam, and F. Gong, FEMS Microbiol. Lett. 134:299–307, 1995). A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified. Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA. Strain 30-84.A2 was not complemented by
gacA
or any cosmid from a genomic library of strain 30-84 but was complemented by
gacS
(formerly
lemA
) homologs from
Pseudomonas fluorescens
Pf-5 (N. Corbel and J. E. Loper, J. Bacteriol. 177:6230–6236, 1995) and
Pseudomonas syringae
pv. syringae B728a (E. M. Hrabek and D. K. Willis, J. Bacteriol. 174:3011–3020, 1992). Transcription of
phzR
was not altered in either mutant; however,
phzI
transcription was eliminated in strains 30-84W and 30-84.A2. These results indicated that the GacS/GacA two-component signal transduction system of
P. aureofaciens
30-84 controls the production of AHL required for phenazine production by mediating the transcription of
phzI
. Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels. Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact.
Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil
Authors: Chidthaisong Amnat , Rosenstock Bernd , Conrad Ralf
Published: December 2019

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-
14
C]glucose and [2-
14
C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-
14
C]glucose released [
14
C]acetate and
14
CO
2
as the main products, with [
14
C]acetate being released faster than
14
CO
2
. The products of [2-
14
C]acetate metabolism, on the other hand, were
14
CO
2
during the reduction phase of soil incubation (days 0 to 15) and
14
CH
4
during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH
4
was produced from acetate, of which up to 56% was produced from glucose degradation.
Multiple Epoxide Hydrolases in
Alternaria alternata
f. sp.
lycopersici
and Their Relationship to Medium Composition and Host-Specific Toxin Production
Authors: Morisseau Christophe , Ward Barney L. , Gilchrist David G. , Hammock Bruce D.
Published: December 2019

The production of
Alternaria alternata
f. sp.
lycopersici
host-specific toxins (AAL toxins) and epoxide hydrolase (EH) activity were studied during the growth of this plant-pathogenic fungus in stationary liquid cultures. Media containing pectin as the primary carbon source displayed peaks of EH activity at day 4 and at day 12. When pectin was replaced by glucose, there was a single peak of EH activity at day 6. Partial characterization of the EH activities suggests the presence of three biochemically distinguishable EH activities. Two of them have a molecular mass of 25 kDa and a pI of 4.9, while the other has a molecular mass of 20 kDa and a pI of 4.7. Each of the EH activities can be distinguished by substrate preference and sensitivity to inhibitors. The EH activities present at day 6 (glucose) or day 12 (pectin) are concomitant with AAL toxin production.
Occurrence of Nontuberculous Mycobacteria in Environmental Samples
Authors: Covert Terry C. , Rodgers Mark R. , Reyes Antolin L. , Stelma Gerard N.
Published: December 2019

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was
M. mucogenicum
(formerly referred to as an
M. chelonae
-like organism).
Role of
pfkA
and General Carbohydrate Catabolism in Seed Colonization by
Enterobacter cloacae
Authors: Roberts D. P. , Dery P. D. , Yucel I. , Buyer J. , Holtman M. A. , Kobayashi D. Y.
Published: December 2019

Enterobacter cloacae
A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128–1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (
P
≤ 0.05) for cucumber and radish seeds compared to that of strain 501R3, but colonization of pea, soybean, sunflower, and sweet corn seeds was not reduced. Pea seeds released several orders of magnitude more total carbohydrates and amino acids than cucumber and radish seeds and approximately 4,000-fold more fructose. Fructose was the only carbohydrate detected in the seed exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released significantly greater amounts of fructose and total carbohydrates and amino acids than cucumber or radish seeds. The exogenous addition of fructose to cucumber and radish seeds at quantities similar to the total quantity of carbohydrates released from pea seeds over 96 h increased the populations of strain A-11 to levels comparable to those of strain 501R3 in sterile sand. Molecular characterization of strain A-11 indicated that the mini-Tn
5
kanamycin transposon was inserted in a region of the genome with significant homology to
pfkA
, which encodes phosphofructo kinase. A comparison of strain A-11 with
Escherichia coli
DF456, a known
pfkA
mutant, indicated that the nutritional loss phenotypes were identical. Furthermore, the
pfkA
homolog cloned from
E. cloacae
501R3 complemented the nutritional loss phenotypes of both
E. coli
DF456 and
E. cloacae
A-11 and restored colonization by strain A-11 to near wild-type levels. These genetic and biochemical restoration experiments provide strong evidence that the quantities of reduced carbon sources found in seed exudates and the ability of microbes to use these compounds play important roles in the colonization of the spermosphere.
Generation of a Superoxide Dismutase (SOD)-Deficient Mutant of
Campylobacter coli
: Evidence for the Significance of SOD in Campylobacter Survival and Colonization
Authors: Purdy Desmond , Cawthraw Sean , Dickinson Joanne H. , Newell Diane G. , Park Simon F.
Published: December 2019

The microaerophilic nature of
Campylobacter
species implies an inherent sensitivity towards oxygen and its reduction products, particularly the superoxide anion. The deleterious effects of exposure to superoxide radicals are counteracted by the activity of superoxide dismutase (SOD). We have shown previously that
Campylobacter coli
possesses an iron cofactored SOD. The
sodB
gene of
C. coli
UA585 was insertionally inactivated by the site-specific insertion of a
tetO
cassette. Organisms harboring the inactivated gene failed to produce a biologically functional form of the enzyme. While the ability of this mutant to grow in aerobic conditions was unchanged relative to the parental strain, its survival was severely compromised when nongrowing cells were exposed to air. Accordingly, the SOD-deficient mutant was unable to survive for prolonged periods in model foods. Furthermore, inactivation of the
sodB
gene decreased the colonization potential in an experimental infection of 1-day-old chicks. In contrast, strain CK100, which is deficient in catalase activity, showed the same survival and colonization characteristics as the parental strain. These results indicate that SOD, but not catalase, is an important determinant in the ability of
C. coli
to survive aerobically and for optimal colonization within the chicken gut.