bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Spore Coat Layers in
Bacillus subtilis
Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation
Authors: Riesenman Paul J. , Nicholson Wayne L. 
Published: July 2002

ABSTRACT

Spores of
Bacillus subtilis
possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of
B. subtilis
(containing the
gerE36
and/or
cotE::cat
mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H
2
O
2
) and various artificial and solar UV treatments. Spores of strains carrying mutations in
gerE
and/or
cotE
were very sensitive to lysozyme and to 5% H
2
O
2
, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the
gerE36
mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the
cotE::cat
mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the
gerE36
and
cotE::cat
mutations behaved like
gerE36
mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.
Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in
Escherichia coli
Authors: Nishihara Kazuyo , Kanemori Masaaki , Yanagi Hideki , Yura Takashi 
Published: July 2002

ABSTRACT

To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in
Escherichia coli
, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in
E. coli
.
Sequencing and Expression of Additional Xylanase Genes from the Hyperthermophile
Thermotoga maritima
FjSS3B.1
Authors: Reeves Rosalind A. , Gibbs Moreland D. , Morris Daniel D. , Griffiths Katherine R. , Saul David J. , Bergquist Peter L. 
Published: July 2002

ABSTRACT

Two genes,
xynB
and
xynC
, coding for xylanases were isolated from
Thermotoga maritima
FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to
xynB
,
xynC
appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.
In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from
Acinetobacter johnsonii
210A
Authors: Resnick Sol M. , Zehnder Alexander J. B. 
Published: July 2002

ABSTRACT

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from
Acinetobacter johnsonii
strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP

n

+ AMP → ADP + polyP

n
−1

. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in
A. johnsonii
210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of
A. johnsonii
210A cell extract, MgCl
2
, polyP

n
=35

, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from
A. johnsonii
is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.
Bacterial Growth Stimulation with Exogenous Siderophore and Synthetic
N
-Acyl Homoserine Lactone Autoinducers under Iron-Limited and Low-Nutrient Conditions
Authors: Guan Le Luo , Onuki Hiroyuki , Kamino Kei 
Published: July 2002

ABSTRACT

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for
Pelagiobacter
sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 μM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM
N
-acyl-octanoylhomoserine lactone (C
8
-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C
8
-HSL; however, C
8
-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.
Influence of Salts on Virus Adsorption to Microporous Filters
Authors: Lukasik Jerzy , Scott Troy M. , Andryshak Diane , Farrah Samuel R. 
Published: July 2002

ABSTRACT

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl
2
, and AlCl
3
) on the adsorption of several viruses (MS2, PRD-1, φX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).
Spatial and Temporal Variations in Chitinolytic Gene Expression and Bacterial Biomass Production during Chitin Degradation
Authors: Baty Ace M. , Eastburn Callie C. , Techkarnjanaruk Somkiet , Goodman Amanda E. , Geesey Gill G. 
Published: July 2002

ABSTRACTGrowth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day−1were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day−1on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading geneschiAandchiB. Cells displaying high levels ofchiA-chiBexpression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population.
Dissolution of Xylose Metabolism in
Lactococcus lactis
Authors: Erlandson Karn A. , Park Joo-Heon , Wissam  , El Khal  , Kao Hsin-Hsin , Basaran Pervin , Brydges Susannah , Batt Carl A. 
Published: July 2002

ABSTRACT

Xylose metabolism, a variable phenotype in strains of
Lactococcus lactis
, was studied and evidence was obtained for the accumulation of mutations that inactivate the
xyl
operon. The xylose metabolism operon (
xylRAB
) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the
xyl
locus were sequenced from
L. lactis
subsp.
lactis
B-4449 (formerly
Lactobacillus xylosus
),
L. lactis
subsp.
lactis
IO-1, and
L. lactis
subsp.
lactis
210, respectively. The two environmental isolates,
L. lactis
B-4449 and
L. lactis
IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose.
L. lactis
210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl
−
. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl
+
strains. Sequence comparisons revealed a number of point mutations in the
xylA
,
xylB
, and
xylR
genes in
L. lactis
210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in
xylB
, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of
L. lactis
unable to metabolize xylose.
Investigation of Candidate Division TM7, a Recently Recognized Major Lineage of the Domain
Bacteria
with No Known Pure-Culture Representatives
Authors: Hugenholtz Philip , Tyson Gene W. , Webb Richard I. , Wagner Ankia M. , Blackall Linda L. 
Published: July 2002

ABSTRACT

A molecular approach was used to investigate a recently described candidate division of the domain
Bacteria
, TM7, currently known only from environmental 16S ribosomal DNA sequence data. A number of TM7-specific primers and probes were designed and evaluated. Fluorescence in situ hybridization (FISH) of a laboratory scale bioreactor using two independent TM7-specific probes revealed a conspicuous sheathed-filament morphotype, fortuitously enriched in the reactor. Morphologically, the filament matched the description of the Eikelboom morphotype 0041-0675 widely associated with bulking problems in activated-sludge wastewater treatment systems. Transmission electron microscopy of the bioreactor sludge demonstrated that the sheathed-filament morphotype had a typical gram-positive cell envelope ultrastructure. Therefore, TM7 is only the third bacterial lineage recognized to have gram-positive representatives. TM7-specific FISH analysis of two full-scale wastewater treatment plant sludges, including the one used to seed the laboratory scale reactor, indicated the presence of a number of morphotypes, including sheathed filaments. TM7-specific PCR clone libraries prepared from the two full-scale sludges yielded 23 novel TM7 sequences. Three subdivisions could be defined based on these data and publicly available sequences. Environmental sequence data and TM7-specific FISH analysis indicate that members of the TM7 division are present in a variety of terrestrial, aquatic, and clinical habitats. A highly atypical base substitution (
Escherichia coli
position 912; C to U) for bacterial 16S rRNAs was present in almost all TM7 sequences, suggesting that TM7 bacteria, like
Archaea
, may be streptomycin resistant at the ribosome level.
Cloning and Characterization of a Periplasmic Nuclease of
Vibrio vulnificus
and Its Role in Preventing Uptake of Foreign DNA
Authors: Wu Shi-I , Lo Shi-Kan , Shao Chung-Ping , Tsai Hsing-Wen , Hor Lien-I 
Published: July 2002

ABSTRACT

We have cloned a nuclease gene,
vvn
, from
Vibrio vulnificus
, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either
V. vulnificus
or recombinant
Escherichia coli
strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in
E. coli
DH5α reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient
V. vulnificus
mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either
E. coli
or
V. vulnificus
. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of
V. vulnificus
mice.
Direct Cloning from Enrichment Cultures, a Reliable Strategy for Isolation of Complete Operons and Genes from Microbial Consortia
Authors: Entcheva Plamena , Liebl Wolfgang , Johann Andre , Hartsch Thomas , Streit Wolfgang R. 
Published: July 2002

ABSTRACT

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the
Escherichia coli
biotin auxotrophic strain ATCC 33767 Δ(
bio-uvrB
). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to
bio
operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (
hut
), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (
moaABCDE
).
Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining
Authors: Grégori Gérald , Citterio Sandra , Ghiani Alessandra , Labra Massimo , Sgorbati Sergio , Brown Spencer , Denis Michel 
Published: July 2002

ABSTRACT
The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
Pathogenicity of
Moraxella osloensis
, a Bacterium Associated with the Nematode
Phasmarhabditis hermaphrodita
, to the Slug
Deroceras reticulatum
Authors: Tan Li , Grewal Parwinder S. 
Published: July 2002

ABSTRACT

Moraxella osloensis
, a gram-negative bacterium, is associated with
Phasmarhabditis hermaphrodita
, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug,
Deroceras reticulatum
. Infective juveniles of
P. hermaphrodita
invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that
M. osloensis
alone is pathogenic to
D. reticulatum
after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of
P. hermaphrodita
to
D. reticulatum
result from the loss of
M. osloensis
from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of
D. reticulatum
in the nematode-bacterium complex and that
P. hermaphrodita
acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by
M. osloensis
is being pursued.