bluuebunny/crossref_metadata_2025 huggingface dataset:
Lethality of a Heat- and Phosphate-Catalyzed Glucose By-Product to
Escherichia coli
O157:H7 and Partial Protection Conferred by the
rpoS
Regulon
Authors: Byrd Jeffrey J. , Cheville Ann M. , Bose Jeffrey L. , Kaspar Charles W.
Published: December 2019

A by-product of glucose produced during sterilization (121°C, 15 lb/in
2
, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to
Escherichia coli
O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25°C. An
rpoS
mutant (pRR10::
rpoS
) of strain 43895 (FRIK 816-3) was significantly (
P
< 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log
10
reduction in CFU per milliliter in 2 days at 25°C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (
P
< 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of
rpoS
and the regulation of
rpoS
-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4°C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to
Salmonella typhimurium
,
Shigella dysenteriae
, and a
Klebsiella
sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to
E. coli
O157:H7 and the protective effects afforded by
rpoS
-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.
Vertical Distribution of Methanogens in the Anoxic Sediment of Rotsee (Switzerland)
Authors: Zepp Falz K. , Holliger C. , Großkopf R. , Liesack W. , Nozhevnikova A. N. , Müller B. , Wehrli B. , Hahn D.
Published: December 2019

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those ofMethanosaetaspp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont ofPlagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domainsBacteriaandArchaeaand the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 ± 0.25) × 1010to (2.62 ± 0.58) × 1010cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domainsBacteriaandArchaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells asMethanosaetaspp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont ofP. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [14C]acetate or [14C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclasticMethanosaetaspp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont ofP. nasuta, accounting on average for 7% of the archaeal population, were also detected.
Use of Antibacterial Agents To Elucidate the Etiology of Juvenile Oyster Disease (JOD) in
Crassostrea virginica
and Numerical Dominance of an α-Proteobacterium in JOD-Affected Animals
Authors: Boettcher Katherine J. , Barber Bruce J. , Singer John T.
Published: December 2019

Since 1988, juvenile oyster disease (JOD) has resulted in high seasonal losses of cultured Eastern oysters (
Crassostrea virginica
) in the Northeast. Although the cause of JOD remains unknown, most evidence is consistent with either a bacterial or a protistan etiology. For the purpose of discerning between these hypotheses, the antibacterial antibiotics norfloxacin and sulfadimethoxine-ormetoprim (Romet-B) were tested for the ability to delay the onset of JOD mortality and/or reduce the JOD mortality of cultured juvenile
C. virginica
. Hatchery-produced
C. virginica
seed were exposed in triplicate groups of 3,000 animals each to either norfloxacin, sulfadimethoxine-ormetoprim, or filter-sterilized seawater (FSSW) and deployed in floating trays on the Damariscotta River of Maine on 17 July 1997. Each week thereafter, a subset of animals from each group was reexposed to the assigned treatment. Repeated immersion in either a sulfadimethoxine-ormetoprim or a norfloxacin solution resulted in a delay in the onset of JOD mortality in treated animals and reduced weekly mortality rates. Weekly treatments with either norfloxacin or sulfadimethoxine-ormetoprim also resulted in a statistically significant reduction in cumulative mortality (55 and 67% respectively) compared to animals treated weekly with FSSW (81%) or those that had received only a single treatment with either norfloxacin, sulfadimethoxine-ormetoprim, or FSSW (77, 84, and 82%, respectively). Bacteriological analyses revealed a numerically dominant bacterium in those animals with obvious signs of JOD. Sequence analysis of the 16S rRNA gene from these bacteria indicates that they are a previously undescribed species of marine α-proteobacteria.
Characterization of
Pseudomonas aeruginosa
Bacteriophage UNL-1, a Bacterial Virus with a Novel UV-A-Inducible DNA Damage Reactivation Phenotype
Authors: Shaffer Julie J. , Jacobsen Lisa M. , Schrader John O. , Lee Kit W. , Martin Eugene L. , Kokjohn Tyler A.
Published: December 2019

UNL-1, a lytic virus of
Pseudomonas aeruginosa
, was observed to express a novel inducible DNA damage reactivation activity in UV-A-irradiated
P. aeruginosa
host cells. The expression of bacteriophage reactivation was quantified in hosts exposed to either UV-C or UV-A radiation. While reactivation of UV-C-damaged UNL-1 was not inducible in UV-C-irradiated host cells, an approximately 13-fold induction was observed in UV-A-irradiated host cells. When host cells were exposed to sunlight, reactivation of damaged UNL-1 virus increased eightfold. The UV-A induction of UNL-1 DNA damage reactivation was supported in hosts lacking
recA
gene function. This report is the first description of a
recA
-independent, UV-inducible virus DNA damage repair system. Our findings suggest that a combination of both host and virus DNA repair processes contribute to the persistence and sustained replication of some bacterial viruses in aquatic environments.
Enhancement of Solubilization and Biodegradation of Polyaromatic Hydrocarbons by the Bioemulsifier Alasan
Authors: Barkay T. , Navon-Venezia S. , Ron E. Z. , Rosenberg E.
Published: December 2019

Alasan, a high-molecular-weight bioemulsifier complex of an anionic polysaccharide and proteins that is produced by
Acinetobacter radioresistens
KA53 (S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E. Z. Ron, and E. Rosenberg, Appl. Environ. Microbiol. 61:3240–3244, 1995), enhanced the aqueous solubility and biodegradation rates of polyaromatic hydrocarbons (PAHs). In the presence of 500 μg of alasan ml
−1
, the apparent aqueous solubilities of phenanthrene, fluoranthene, and pyrene were increased 6.6-, 25.7-, and 19.8-fold, respectively. Physicochemical characterization of the solubilization activity suggested that alasan solubilizes PAHs by a physical interaction, most likely of a hydrophobic nature, and that this interaction is slowly reversible. Moreover, the increase in apparent aqueous solubility of PAHs does not depend on the conformation of alasan and is not affected by the formation of multimolecular aggregates of alasan above its saturation concentration. The presence of alasan more than doubled the rate of [
14
C]fluoranthene mineralization and significantly increased the rate of [
14
C]phenanthrene mineralization by
Sphingomonas paucimobilis
EPA505. The results suggest that alasan-enhanced solubility of hydrophobic compounds has potential applications in bioremediation.
Development of a Self-Cloning System for
Actinomadura verrucosospora
and Identification of Polyketide Synthase Genes Essential for Production of the Angucyclic Antibiotic Pradimicin
Authors: Dairi Tohru , Hamano Yoshimitsu , Furumai Tamotsu , Oki Toshikazu
Published: December 2019

A self-cloning system for
Actinomadura verrucosospora
, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for
A. verrucosospora
and a novel plasmid vector that consists of a replicon from a newly found
Actinomadura
plasmid and a selectable marker cloned from the
Actinomadura
strain. The system has an efficiency of more than 10
5
CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from
A. verrucosospora
. Nucleotide sequence analysis of the 3.5-kb
Sal
I-
Sph
I fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.
Occurrence, Diversity, and Pathogenicity of Halophilic
Vibrio
spp. and Non-O1
Vibrio cholerae
from Estuarine Waters along the Italian Adriatic Coast
Authors: Barbieri Elena , Falzano Loredana , Fiorentini Carla , Pianetti Anna , Baffone Wally , Fabbri Alessia , Matarrese Paola , Casiere Annarita , Katouli Mohammad , Kühn Inger , Möllby Roland , Bruscolini Francesca , Donelli Gianfranco
Published: December 2019

The occurrence, diversity, and pathogenicity of
Vibrio
spp. were investigated in two estuaries along the Italian Adriatic coast.
Vibrio alginolyticus
was the predominant species, followed by
Vibrio parahaemolyticus
, non-O1
Vibrio cholerae
, and
Vibrio vulnificus
. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of
Vibrio
strains. These findings indicate a significant presence of potentially pathogenic
Vibrio
strains along the Adriatic coast.
Specificity of
Lactococcus lactis
subsp.
cremoris
SK11 Proteinase, Lactocepin III, in Low-Water-Activity, High-Salt-Concentration Humectant Systems and Its Stability Compared with That of Lactocepin I
Authors: Reid Julian R. , Coolbear Tim
Published: December 2019

Marked changes in the specificity of hydrolysis of α
s1
-, β-, and κ-caseins by lactocepin III from
Lactococcus lactis
subsp.
cremoris
SK11 were found in humectant systems giving the equivalent water activity (a
w
) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from
L. lactis
subsp.
cremoris
HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, a
w
, pH, and NaCl concentration was complex. Nevertheless, in those systems where a
w
, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.
Visualization and Modelling of the Thermal Inactivation of Bacteria in a Model Food
Authors: Bellara Sanjay R. , Fryer Peter J. , McFarlane Caroline M. , Thomas Colin R. , Hocking Paul M. , Mackey Bernard M.
Published: December 2019

A large number of incidents of food poisoning have been linked to undercooked meat products. The use of mathematical modelling to describe heat transfer within foods, combined with data describing bacterial thermal inactivation, may prove useful in developing safer food products while minimizing thermal overprocessing. To examine this approach, cylindrical agar blocks containing immobilized bacteria (
Salmonella typhimurium
and
Brochothrix thermosphacta
) were used as a model system in this study. The agar cylinders were subjected to external conduction heating by immersion in a water bath. They were then incubated, sliced open, and examined by image analysis techniques for regions of no bacterial growth. A finite-difference scheme was used to model thermal conduction and the consequent bacterial inactivation. Bacterial inactivation rates were modelled with values for the time required to reduce bacterial number by 90% (D) and the temperature increase required to reduce D by 90% taken from the literature. Model simulation results agreed well with experimental results for both bacteria, demonstrating the utility of the technique.
Identification and Characterization of a Flagellin Gene from the Endosymbiont of the Hydrothermal Vent Tubeworm
Riftia pachyptila
Authors: Millikan Deborah S. , Felbeck Horst , Stein Jeffrey L.
Published: December 2019

The bacterial endosymbionts of the hydrothermal vent tubeworm
Riftia pachyptila
play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment. To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont
fliC
gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus ς
28
RNA polymerase recognition site lay upstream of the proposed translational start site. The symbiont protein was expressed in
Escherichia coli
, and flagella were observed by electron microscopy. A 30,000-
M
r
protein subunit was identified in whole-cell extracts by Western blot analysis. These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of
R. pachyptila
is acquired with each new host generation.
Phylogenetic Analysis of Particle-Attached and Free-Living Bacterial Communities in the Columbia River, Its Estuary, and the Adjacent Coastal Ocean
Authors: Crump Byron C. , Armbrust E. Virginia , Baross John A.
Published: December 2019

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class
Proteobacteria
. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus
Cytophaga
or of the α, γ, or δ subclass of the class
Proteobacteria
. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (β-proteobacteria, gram-positive bacteria, and
Verrucomicrobium
spp.) and groups of marine organisms (gram-positive bacteria and α-proteobacteria [SAR11 and
Rhodobacter
spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.
Purification, Characterization, and Heterologous Expression in
Fusarium venenatum
of a Novel Serine Carboxypeptidase from
Aspergillus oryzae
Authors: Blinkovsky Alexander M. , Byun Tony , Brown Kimberly M. , Golightly Elizabeth J.
Published: December 2019

A novel serine carboxypeptidase (EC
3.4.16.1
) was found in an
Aspergillus oryzae
fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25°C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60°C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform a
Fusarium venenatum
host strain. The transformed strain of
F. venenatum
expressed an active recombinant carboxypeptidase. In
F. venenatum
, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from
A. oryzae
. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.
Genetic Structure of Natural Populations of
Escherichia coli
in Wild Hosts on Different Continents
Authors: Souza Valeria , Rocha Martha , Valera Aldo , Eguiarte Luis E.
Published: December 2019

Current knowledge of genotypic and phenotypic diversity in the species
Escherichia coli
is based almost entirely on strains recovered from humans or zoo animals. In this study, we analyzed a collection of 202 strains obtained from 81 mammalian species representing 39 families and 14 orders in Australia and the Americas, as well as several reference strains; we also included a strain from a reptile and 10 from different families of birds collected in Mexico. The strains were characterized genotypically by multilocus enzyme electrophoresis (MLEE) and phenotypically by patterns of sugar utilization, antibiotic resistance, and plasmid profile. MLEE analysis yielded an estimated genetic diversity (H) of 0.682 for 11 loci. The observed genetic diversity in this sample is the greatest yet reported for
E. coli
. However, this genetic diversity is not randomly distributed; geographic effects and host taxonomic group accounted for most of the genetic differentiation. The genetic relationship among the strains showed that they are more associated by origin and host order than is expected by chance. In a dendrogram, the ancestral cluster includes primarily strains from Australia and ECOR strains from groups B and C. The most differentiated
E. coli
in our analysis are strains from Mexican carnivores and strains from humans, including those in the ECOR group A. The kinds and numbers of sugars utilized by the strains varied by host taxonomic group and country of origin. Strains isolated from bats were found to exploit the greatest range of sugars, while those from primates utilized the fewest. Toxins are more frequent in strains from rodents from both continents than in any other taxonomic group. Strains from Mexican wild mammals were, on average, as resistant to antibiotics as strains from humans in cities. On average, the Australian strains presented a lower antibiotic resistance than the Mexican strains. However, strains recovered from hosts in cities carried significantly more plasmids than did strains isolated from wild mammals. Previous studies have shown that natural populations of
E. coli
harbor an extensive genetic diversity that is organized in a limited number of clones. However, knowledge of this worldwide bacterium has been limited. Here, we suggest that the strains from a wide range of wild hosts from different regions of the world are organized in an ecotypic structure where adaptation to the host plays an important role in the population structure.
Heat-Induced Expression and Chemically Induced Expression of the
Escherichia coli
Stress Protein HtpG Are Affected by the Growth Environment
Authors: Mason C. Anthony , Dünner Janine , Indra Paul , Colangelo Teresa
Published: December 2019

Differences in expression of the
Escherichia coli
stress protein HtpG were found following exposure of exponentially growing cells to heat or chemical shock when cells were grown under different environmental conditions. With an
htpG::lacZ
reporter system,
htpG
expression increased in cells grown in a complex medium (Luria-Bertani [LB] broth) following a temperature shock at 45°C. In contrast, no HtpG overexpression was detected in cells grown in a glucose minimal medium, despite a decrease in the growth rate. Similarly, in pyruvate-grown cells there was no heat shock induction of HtpG expression, eliminating the possibility that repression of HtpG in glucose-grown
E. coli
was due to catabolite repression. When 5 mM phenol was used as a chemical stress agent for cells growing in LB broth, expression of HtpG increased. However, when LB-grown cells were subjected to stress with 10 mM phenol and when both 5 and 10 mM phenol were added to glucose-grown cultures, repression of
htpG
expression was observed. 2-Chlorophenol stress resulted in overexpression of HtpG when cells were grown in complex medium but repression of HtpG synthesis when cells were grown in glucose. No induction of
htpG
expression was seen with 2,4-dichlorophenol in cells grown with either complex medium or glucose. The results suggest that, when a large pool of amino acids and proteins is available, as in complex medium, a much stronger stress response is observed. In contrast, when cells are grown in a simple glucose mineral medium,
htpG
expression either is unaffected or is even repressed by imposition of a stress condition. The results demonstrate the importance of considering differences in growth environment in order to better understand the nature of the response to an imposed stress condition.
Community Analysis of Biofilters Using Fluorescence In Situ Hybridization Including a New Probe for the
Xanthomonas
Branch of the Class
Proteobacteria
Authors: Friedrich Udo , Naismith Michèle M. , Altendorf Karlheinz , Lipski André
Published: December 2019

Domain-, class-, and subclass-specific rRNA-targeted probes were applied to investigate the microbial communities of three industrial and three laboratory-scale biofilters. The set of probes also included a new probe (named XAN818) specific for the
Xanthomonas
branch of the class
Proteobacteria
; this probe is described in this study. The members of the
Xanthomonas
branch do not hybridize with previously developed rRNA-targeted oligonucleotide probes for the α-, β-, and γ-
Proteobacteria
. Bacteria of the
Xanthomonas
branch accounted for up to 4.5% of total direct counts obtained with 4′,6-diamidino-2-phenylindole. In biofilter samples, the relative abundance of these bacteria was similar to that of the γ-
Proteobacteria. Actinobacteria
(gram-positive bacteria with a high G+C DNA content) and α-
Proteobacteria
were the most dominant groups. Detection rates obtained with probe EUB338 varied between about 40 and 70%. For samples with high contents of gram-positive bacteria, these percentages were substantially improved when the calculations were corrected for the reduced permeability of gram-positive bacteria when formaldehyde was used as a fixative. The set of applied bacterial class- and subclass-specific probes yielded, on average, 58.5% (± a standard deviation of 23.0%) of the corrected eubacterial detection rates, thus indicating the necessity of additional probes for studies of biofilter communities. The
Xanthomonas
-specific probe presented here may serve as an efficient tool for identifying potential phytopathogens. In situ hybridization proved to be a practical tool for microbiological studies of biofiltration systems.
Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (
phaC
) Gene from
Chromobacterium violaceum
Authors: Kolibachuk Dana , Miller Andrea , Dennis Douglas
Published: December 2019

The polyhydroxyalkanoic acid synthase gene from
Chromobacterium violaceum
(
phaC
Cv
) was cloned and characterized. A 6.3-kb
Bam
HI fragment was found to contain both
phaC
Cv
and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (
phaA
Cv
).
Escherichia coli
strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While
C. violaceum
accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) when grown on a fatty acid carbon source,
Klebsiella aerogenes
and
Ralstonia eutropha
(formerly
Alcaligenes eutrophus
), harboring
phaC
Cv
, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-
co
-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the
C. violaceum
PHA synthase. Neither recombinant
E. coli
nor recombinant
Pseudomonas putida
harboring
phaC
Cv
accumulated significant levels of PHA. Sequence analysis of the
phaC
Cv
product shows homology with several PHA synthases, most notably a 48% identity with that of
Alcaligenes latus
(GenBank accession no. AAD10274).
Diel Rhythms in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Glutamine Synthetase Gene Expression in a Natural Population of Marine Picoplanktonic Cyanobacteria (
Synechococcus
spp.)
Authors: Wyman Michael
Published: December 2019

Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine
Synechococcus
spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean.
Synechococcus
sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in
cpeBA
mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (
rbcL
) and glutamine synthetase (
glnA
) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of
rbcL
transcription was clearly not light dependent, expression of
glnA
appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine
Synecoccoccus
spp.
Quantification of the Flavonoid-Degrading Bacterium
Eubacterium ramulus
in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe
Authors: Simmering Rainer , Kleessen Brigitta , Blaut Michael
Published: December 2019

To investigate the occurrence of the flavonoid-degrading bacterium
Eubacterium ramulus
in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of
E. ramulus
in each individual tested in numbers from 4.4 × 10
7
to 2.0 × 10
9
cells/g of fecal dry mass. Six fecal
E. ramulus
isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.
Bacterioplankton Compositions of Lakes and Oceans: a First Comparison Based on Fluorescence In Situ Hybridization
Authors: Glöckner Frank Oliver , Fuchs Bernhard M. , Amann Rudolf
Published: December 2019

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains
Bacteria
and
Archaea
, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the
Cytophaga-Flavobacterium
cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4′,6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of
Proteobacteria
as well as members of the
Planctomycetales
were detected in both freshwater and marine water in abundances <7%.
Physicochemical Parameters for Growth of the Sea Ice Bacteria
Glaciecola punicea
ACAM 611
T
and
Gelidibacter
sp. Strain IC158
Authors: Nichols D. S. , Greenhill A. R. , Shadbolt C. T. , Ross T. , McMeekin T. A.
Published: December 2019

The water activity and pH ranges for growth of
Glaciecola punicea
(a psychrophile) were extended when this organism was grown at suboptimal rather than optimal temperatures. No such extension was observed for
Gelidibacter
sp. strain IC158 (a psychrotolerant bacterium) at analogous temperatures. Salinity and pH may be primary physicochemical parameters controlling bacterial community development in sea ice.
Polyphosphate Kinase of
Acinetobacter
sp. Strain ADP1: Purification and Characterization of the Enzyme and Its Role during Changes in Extracellular Phosphate Levels
Authors: Trelstad Piper L. , Purdhani Pooja , Geißdörfer Walter , Hillen Wolfgang , Keasling J. D.
Published: December 2019

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of
Acinetobacter
sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl
2
. The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40°C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per μg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in
Acinetobacter
sp. strain ADP1. Under low-phosphate (P
i
) conditions, despite strong induction of the
ppk
gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in
Acinetobacter
sp. strain ADP1. Once excess phosphate was added to the P
i
-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P
i
conditions. This activity declined when phosphate was added.
Development and Characterization of a Gene Expression Reporter System for
Clostridium acetobutylicum
ATCC 824
Authors: Tummala Seshu B. , Welker Neil E. , Papoutsakis Eleftherios T.
Published: December 2019

A gene expression reporter system (pHT3) for
Clostridium acetobutylicum
ATCC 824 was developed by using the
lacZ
gene from
Thermoanaerobacterium thermosulfurogenes
EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes,
ptb
(coding for phosphotransbutyrylase),
thl
(coding for thiolase), and
adc
(coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in
C. acetobutylicum
. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the
lacZ
gene from
T. thermosulfurogenes
EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the
ptb
promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.
Generalized Transduction of Small
Yersinia enterocolitica
Plasmids
Authors: Hertwig Stefan , Popp Andreas , Freytag Barbara , Lurz Rudi , Appel Bernd
Published: December 2019

To study phage-mediated gene transfer in
Yersinia
, the ability of
Yersinia
phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic
Yersinia enterocolitica
strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10
−5
to 10
−7
/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic
Yersinia
strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes.
Purification of Mutacin III from Group III
Streptococcus mutans
UA787 and Genetic Analyses of Mutacin III Biosynthesis Genes
Authors: Qi Fengxia , Chen Ping , Caufield Page W.
Published: December 2019

Previously, members of our group reported the isolation and characterization of mutacin II from
Streptococcus mutans
T8 and the genetic analyses of the mutacin II biosynthesis genes (J. Novak, P. W. Caufield, and E. J. Miller, J. Bacteriol. 176:4316–4320, 1994; F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:652–658, 1999; P. Chen, F. Qi, J. Novak, and P. W. Caufield, Appl. Environ. Microbiol. 65:1356–1360, 1999). In this study, we cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of
S. mutans
, UA787. DNA sequence analysis revealed eight open reading frames, which we designated
mutR
,
-A
,
-A′
,
-B
,
-C
,
-D
,
-P
, and
-T
. MutR bears strong homology with MutR of mutacin II, while MutA, -B, -C, -D, -P, and -T are counterparts of proteins in the lantibiotic epidermin group. MutA′ has 60% amino acid identity with MutA and therefore appears to be a duplicate of MutA. Insertional inactivation demonstrated that
mutA
is an essential gene for mutacin III production, while
mutA′
is not required. Mutacin III was purified to homogeneity by using reverse-phase high-pressure liquid chromatography. N-terminal peptide sequencing of the purified mutacin III determined
mutA
to be the structural gene for prepromutacin III. The molecular mass of the purified peptide was measured by laser disorption mass spectrophotometry and found to be 2,266.43 Da, consistent with our supposition that mutacin III has posttranslational modifications similar to those of the lantibiotic epidermin.
An α-
l
-Arabinofuranosidase from
Trichoderma reesei
Containing a Noncatalytic Xylan-Binding Domain
Authors: Nogawa Masahiro , Yatsui Kenji , Tomioka Akiko , Okada Hirofumi , Morikawa Yasushi
Published: December 2019

l
-Sorbose, an excellent cellulase and xylanase inducer from
Trichoderma reesei
PC-3-7, also induced α-
l
-arabinofuranosidase (α-AF) activity. An α-AF induced by
l
-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for
p
-nitrophenyl-α-
l
-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.
Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from
Cellulomonas pachnodae
Authors: Cazemier Anne E. , Verdoes Jan C. , van Ooyen Albert J. J. , Op den Camp Huub J. M.
Published: December 2019

Two xylanase-encoding genes, named
xyn11A
and
xyn10B
, were isolated from a genomic library of
Cellulomonas pachnodae
by expression in
Escherichia coli
. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology.
C. pachnodae
, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous
Pachnoda marginata
larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from
Cellulomonas fimi
, distinct differences in the molecular organizations of the xylanases from the two
Cellulomonas
species were identified.
Synergistic Actions of Nisin, Sublethal Ultrahigh Pressure, and Reduced Temperature on Bacteria and Yeast
Authors: ter Steeg Pieter F. , Hellemons Johan C. , Kok Anja E.
Published: December 2019

Nisin in combination with ultrahigh-pressure treatment (UHP) showed strong synergistic effects against
Lactobacillus plantarum
and
Escherichia coli
at reduced temperatures (<15°C). The strongest inactivation effects were observed when nisin was present during pressure treatment and in the recovery medium. Elimination (>6-log reductions) of
L. plantarum
was achieved at 10°C with synergistic combinations of 0.5 μg of nisin per ml at 150 MPa and 0.1 μg of nisin per ml at 200 MPa for 10 min. Additive effects of nisin and UHP accounted for only 1.2- and 3.7-log reductions, respectively. Elimination was also achieved for
E. coli
at 10°C with nisin present at 2 μg/ml, and 10 min of pressure at 200 MPa, whereas the additive effect accounted for only 2.6-log reductions. Slight effects were observed even against the yeast
Saccharomyces cerevisiae
with nisin present at 5 μg/ml and with 200 MPa of pressure. Combining nisin, UHP, and lowered temperature may allow considerable reduction in time and/or pressure of UHP treatments. Kill can be complete without the frequently encountered survival tails in UHP processing. The slightly enhanced synergistic kill with UHP at reduced temperatures was also observed for other antimicrobials, the synthetic peptides MB21 and histatin 5. The postulated mode of action was that the reduced temperature and the binding of peptides to the membrane increased the efficacy of UHP treatment. The increases in fatty acid saturation or diphosphatidylglycerol content and the lysylphosphatidyl content of the cytoplasm membrane of
L. plantarum
were correlated with increased susceptibility to UHP and nisin, respectively.
Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of
Xanthomonas hyacinthi
Authors: van Doorn J. , Ojanen-Reuhs T. , Hollinger T. C. , Reuhs B. L. , Schots A. , Boonekamp P. M. , Oudega B.
Published: December 2019

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from
Xanthomonas hyacinthi
and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with
X. hyacinthi
cells or fimbriae and for a negative reaction with
X. translucens
pv. graminis or
Erwinia carotovora
subsp.
carotovora
. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two
X. translucens
pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all
X. hyacinthi
strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other
Xanthomonas
pathovars tested or with 14 other bacterial species from other genera, such as
Erwinia
and
Pseudomonas
, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of
X. hyacinthi
in infected hyacinths.
Identification of Bacterial Species Associated with the Sheep Scab Mite (
Psoroptes ovis
) by Using Amplified Genes Coding for 16S rRNA
Authors: Hogg J. C. , Lehane M. J.
Published: December 2019

This was the first molecular study of the bacterial flora of the sheep scab mite (
Psoroptes ovis
). A sequence analysis of genes coding for 16S rRNA revealed that
Serratia marcescens
and bacteria closely related to
Staphylococcus intermedius
or
Staphylococcus chromogens
and
Alloiococcus otitidis
were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by
P. ovis
.
Clostridium difficile
Cell Attachment Is Modified by Environmental Factors
Authors: Waligora Anne-Judith , Barc Marie-Claude , Bourlioux Pierre , Collignon Anne , Karjalainen Tuomo
Published: December 2019

Adherence of
Clostridium difficile
to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.
Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes
Authors: O’Farrell Katrina A. , Janssen Peter H.
Published: December 2019

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division
Verrucomicrobia
in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the
Verrucomicrobia
in DNA extracted from the soil.