bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling
Authors: Schmalenberger Achim , Schwieger Frank , Tebbe Christoph C. 
Published: July 2002

ABSTRACT

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (
Zea mays
), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus
Paenibacillus
(low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.
Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from
Mycobacterium
sp. Strain PYR-1
Authors: Khan Ashraf A. , Wang Rong-Fu , Cao Wei-Wen , Doerge Daniel R. , Wennerstrom David , Cerniglia Carl E. 
Published: July 2002

ABSTRACT

Mycobacterium
sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of
Mycobacterium
sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of
Mycobacterium
sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from
Rhodococcus
spp. and
Nocardioides
spp. The genes from
Mycobacterium
sp. strain PYR-1 were subcloned and overexpressed in
Escherichia coli
with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.
Horizontal Transfer of a Multi-Drug Resistance Plasmid between Coliform Bacteria of Human and Bovine Origin in a Farm Environment
Authors: Oppegaard Hanne , Steinum Terje M. , Wasteson Yngvild 
Published: July 2002

ABSTRACT
Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment.
Development of a Virus Concentration Method and Its Application to Detection of Enterovirus and Norwalk Virus from Coastal Seawater
Authors: Katayama Hiroyuki , Shimasaki Akihiro , Ohgaki Shinichiro 
Published: July 2002

ABSTRACT

We developed a new procedure for concentration of enteric viruses from water using a negatively charged membrane. Rinsing the membrane with 0.5 mM H
2
SO
4
(pH 3.0) in order to elute cations prior to viral elution with 1 mM NaOH (pH 10.5) promoted poliovirus recovery yields from 33 to 95% when applied to pure water and 38 to 89% when applied to natural seawater from Tokyo Bay, Japan, respectively. This method showed average recovery yields of spiked poliovirus of 62% (
n
= 8) from 1 liter of artificial seawater. This method showed higher recovery yields (>61%) than that of the conventional method using positively charged membrane (6%) when applied to seawater. This method is also free from beef extract elution, which has an inhibitory effect in the subsequent viral genome detection by reverse transcription-PCR. Naturally occurring Norwalk viruses from 2 liters of Tokyo Bay water in winter and infectious enteroviruses from 2 liters of recreational coastal seawater in summer were detected by using this viral concentration method.
Characterization of a Chitinase Gene from
Stenotrophomonas maltophilia
Strain 34S1 and Its Involvement in Biological Control
Authors: Kobayashi Donald Y. , Reedy Ralph M. , Bick JulieAnn , Oudemans Peter V. 
Published: July 2002

ABSTRACT

A chitinase gene was cloned on a 2.8-kb DNA fragment from
Stenotrophomonas maltophilia
strain 34S1 by heterologous expression in
Burkholderia cepacia
. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of
S. maltophilia
strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by
chiA
. Marker exchange mutagenesis of
chiA
resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of
S. maltophilia
.
Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters
Authors: Sinton Lester W. , Hall Carollyn H. , Lynch Philippa A. , Davies-Colley Robert J. 
Published: July 2002

ABSTRACT

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci,
Escherichia coli
, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (
k
S
) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (
k
D
) rates in enclosed (control) chambers. The overall
k
S
ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥
E. coli
> somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.
Production of N2through Anaerobic Ammonium Oxidation Coupled to Nitrate Reduction in Marine Sediments
Authors: Thamdrup Bo , Dalsgaard Tage 
Published: July 2002

ABSTRACTIn the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N2, thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N2production in marine sediments. Incubations with15N-labeled nitrate or ammonium demonstrated that during this process, N2is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N2production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N2production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N2production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N2, anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.
Effects of Amendment with Ferrihydrite and Gypsum on the Structure and Activity of Methanogenic Populations in Rice Field Soil
Authors: Lueders Tillmann , Friedrich Michael W. 
Published: July 2002

ABSTRACT

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO
4
· 2H
2
O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 μM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of
Methanosarcina
populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of
Methanosaeta
spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels (∼0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 μM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.
Proteomic Analysis Reveals Differential Protein Expression by
Bacillus cereus
during Biofilm Formation
Authors: Oosthuizen Marinda C. , Steyn Bridgitta , Theron Jacques , Cosette Pascal , Lindsay Denise , von Holy Alexander , Brözel Volker S. 
Published: July 2002

ABSTRACT

Bacillus cereus
, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods.
B. cereus
DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and
l
-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both σ
B
and σ
H
, could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.
Elemental Composition (C, N, P) and Cell Volume of Exponentially Growing and Nutrient-Limited Bacterioplankton
Authors: Vrede Katarina , Heldal Mikal , Norland Svein , Bratbak Gunnar 
Published: July 2002

ABSTRACT

Marine bacterioplankton were isolated and grown in batch cultures until their growth became limited by organic carbon (C), nitrogen (N), or phosphorus (P). Samples were taken from the cultures at both the exponential and stationary phases. The elemental composition of individual bacterial cells was analyzed by X-ray microanalysis with an electron microscope. The cell size was also measured. The elemental content was highest in exponentially growing cells (149 ± 8 fg of C cell
−1
, 35 ± 2 fg of N cell
−1
, and 12 ± 1 fg of P cell
−1
; average of all isolates ± standard error). The lowest C content was found in C-limited cells (39 ± 3 fg of C cell
−1
), the lowest N content in C- and P-limited cells (12 ± 1 and 12 ± 2 fg of N cell
−1
, respectively), and the lowest P content in P-limited cells (2.3 ± 0.6 fg of P cell
−1
). The atomic C:N ratios varied among treatments between 3.8 ± 0.1 and 9.5 ± 1.0 (average ± standard error), the C:P ratios between 35 ± 2 and 178 ± 28, and the N:P ratios between 6.7 ± 0.3 and 18 ± 3. The carbon-volume ratios showed large variation among isolates due to different types of nutrient limitation (from 51± 4 to 241 ± 38 fg of C μm
−1
; average of individual isolates and treatments ± standard error). The results show that different growth conditions and differences in the bacterial community may explain some of the variability of previously reported elemental and carbon-volume ratios.
Description of a 23.9-Kilobase Chromosomal Deletion Containing a Region Encoding
fsr
Genes Which Mainly Determines the Gelatinase-Negative Phenotype of Clinical Isolates of
Enterococcus faecalis
in Urine
Authors: Nakayama Jiro , Kariyama Reiko , Kumon Hiromi 
Published: July 2002

ABSTRACT

Expression of virulence-related extracellular proteases, gelatinase, and serine protease of
Enterococcus faecalis
is regulated by a quorum-sensing system encoded by the
fsr
gene cluster. In this study, a 23.9-kb chromosomal deletion containing the
fsr
gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of
E. faecalis
from urine.
Bacterial Adhesion: Seen Any Good Biofilms Lately?
Authors: Dunne W. Michael 
Published: July 2002

SUMMARY
The process of surface adhesion and biofilm development is a survival strategy employed by virtually all bacteria and refined over millions of years. This process is designed to anchor microorganisms in a nutritionally advantageous environment and to permit their escape to greener pastures when essential growth factors have been exhausted. Bacterial attachment to a surface can be divided into several distinct phases, including primary and reversible adhesion, secondary and irreversible adhesion, and biofilm formation. Each of these phases is ultimately controlled by the expression of one or more gene products. Ultrastructurally, the mature bacterial biofilm resembles an underwater coral reef containing pyramidal or mushroom-shaped microcolonies of organisms embedded within an extracellular glycocalyx, with channels and cavities to allow the exchange of nutrients and waste. The biofilm protects its inhabitants from predators, dehydration, biocides, and other environmental extremes while regulating population growth and diversity through primitive cell signals. From a physiological standpoint, surface-bound bacteria behave quite differently from their planktonic counterparts. Recognizing that bacteria naturally occur as surface-bound and often polymicrobic communities, the practice of performing antimicrobial susceptibility tests using pure cultures and in a planktonic growth mode should be questioned. That this model does not reflect conditions found in nature might help explain the difficulties encountered in the management and treatment of biomedical implant infections.
Effect of α-Oligosaccharide Phenotype of
Neisseria gonorrhoeae
Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells
Authors: Minor Susie Y. , Banerjee Asesh , Gotschlich Emil C. 
Published: July 2002

ABSTRACT
The genes encoding the glycosyltransferases responsible for the addition of the five sugars in the α-oligosaccharide (α-OS) moiety of lipooligosaccharide (LOS) have been identified. Disruption of these glycosyltransferase genes singly or in combination results in corresponding truncations in LOS. In the present work we show that sequential deletion of the terminal four sugar residues of gonococcal α-OS had no discernible effect on the invasion of human conjunctival, endometrial, and cervical cell lines. However, deletion of the proximal glucose, which resulted in the complete deletion of α-OS, significantly impaired invasion of the gonococci into all three cell lines. The effect of deleting α-OS on invasion was independent of and additive to the known invasion-promoting factor OpaA. These data suggest that the proximal glucose residue of the α-OS chain of LOS is required for efficient invasion of gonococci into host mucosa.
Phylogeny of
Vibrio cholerae
Based on
recA
Sequence
Authors: Stine O. Colin , Sozhamannan Shanmuga , Gou Qing , Zheng Siqen , Morris J. Glenn , Johnson Judith A. 
Published: July 2002

ABSTRACT

We sequenced a 705-bp fragment of the
recA
gene from 113
Vibrio cholerae
strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly,
Vibrio parahaemolyticus
and
Vibrio vulnificus
strains formed out-groups; we also identified isolates which resembled
V. cholerae
biochemically but which did not cluster with
V. cholerae
. In many instances,
V. cholerae
serogroup designations did not correlate with phylogeny, as reflected by
recA
sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among
V. cholerae
strains.