bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of High Mutation Rates on the Mechanisms and Dynamics of In Vitro and In Vivo Resistance Development to Single or Combined Antipseudomonal Agents
Authors: Plasencia V. , Borrell N. , Maciá M. D. , Moya B. , Pérez J. L. , Oliver A.
Published: May 2007

We studied the mechanisms and dynamics of the development of resistance to ceftazidime (CAZ) alone or combined with tobramycin (TOB) or ciprofloxacin (CIP) in vitro and in vivo (using a mouse model of lung infection with human antibiotic regimens).Pseudomonas aeruginosastrain PAO1 and its hypermutable derivative PAOΔmutSwere used, and the results were compared with those previously obtained with CIP, TOB, and CIP plus TOB (CIP-TOB) under the same conditions. An important (200-fold) amplification of the number of resistant mutant cells was documented for PAOΔmutS-infected mice that were under CAZ treatment compared to the number for mice that received placebo, whereas the median number of resistant mutant cells was below the detection limits for mice infected by PAO1. These results were intermediate between the high amplification with CIP (50,000-fold) and the low amplification with TOB (10-fold). All CAZ-resistant single mutant cells selected in vitro or in vivo hyperproduced AmpC. On the other hand, the three combinations studied were found to be highly effective in the prevention of in vivo resistance development in mice infected with PAOΔmutS, although the highest therapeutic efficacy (in terms of mortality and total bacterial load reduction) compared to those of the individual regimens was obtained with CIP-TOB and the lowest was with CAZ-CIP. Nevertheless, mutant cells that were resistant to the three combinations tested were readily selected in vitro for PAOΔmutS(mutation rates from 1.2 × 10−9to 5.8 × 10−11) but not for PAO1, highlighting the potential risk for antimicrobial resistance development associated with the presence of hypermutable strains, even when combined therapy was used. All five independent CAZ-TOB-resistant PAOΔmutSdouble mutants studied presented the same resistance mechanism (AmpC hyperproduction plus an aminoglycoside resistance mechanism not related to MexXY), whereas four different combinations of resistance mechanisms were documented for the five CAZ-CIP-resistant double mutants.
Comparative Study of the Effects of Pyridoxine, Rifampin, and Renal Function on Hematological Adverse Events Induced by Linezolid
Authors: Soriano Alex , Ortega Mar , García Sebastián , Peñarroja Georgina , Bové Albert , Marcos Miguel , Martínez Juan C. , Martínez José A. , Mensa Josep
Published: May 2007

Hematological disturbances that develop during linezolid treatment are a major concern when linezolid is administered for prolonged periods of time. The aim of this study was to evaluate the influences of pyridoxine, rifampin, and renal function on hematological adverse events. From January 2002 to April 2006, 52 patients received a long-term course of linezolid. Blood cell counts were monitored weekly. Thrombocytopenia was defined as a decrease to <75% of the baseline platelet count, and anemia was defined when the hemoglobin concentration decreased by ≥2 g/liter from the baseline value. Twenty-four patients received linezolid alone, and 28 patients received linezolid plus 200 mg of pyridoxine. The Kaplan-Meier survival method, followed by the log-rank test, was used to estimate the cumulative probability of adverse events, and Cox regression analysis was performed to evaluate the independent predictors of toxicity. The baseline characteristics of the patients in both groups were similar. The cumulative probability of thrombocytopenia and anemia in patients who received pyridoxine was not different from that in patients who did not receive it. Hematological adverse events were less frequent in patients taking rifampin and were more frequent in patients with renal failure. However; the Cox regression analysis showed that rifampin was the only independent predictor associated with a lower risk of thrombocytopenia (hazard ratio, 0.37; 95% confidence interval, 0.14 to 0.98;P= 0.045). In conclusion, pyridoxine did not prevent linezolid-related hematological adverse events, and the coadministration of rifampin was associated with a lower risk of thrombocytopenia.
Hexadecane and Tween 80 Stimulate Lipase Production in
Burkholderia glumae
by Different Mechanisms
Authors: Boekema Bouke K. H. L. , Beselin Anke , Breuer Michael , Hauer Bernhard , Koster Margot , Rosenau Frank , Jaeger Karl-Erich , Tommassen Jan
Published: April 2007

Burkholderia glumae
strain PG1 produces a lipase of biotechnological relevance. Lipase production by this strain and its derivative LU8093, which was obtained through classical strain improvement, was investigated under different conditions. When 10% hexadecane was included in the growth medium, lipolytic activity in both strains could be increased ∼7-fold after 24 h of growth. Hexadecane also stimulated lipase production in a strain containing the lipase gene fused to the
tac
promoter, indicating that hexadecane did not affect lipase gene expression at the transcriptional level, which was confirmed using
lipA
-
gfp
reporter constructs. Instead, hexadecane appeared to enhance lipase secretion, since the amounts of lipase in the culture supernatant increased in the presence of hexadecane, with a concomitant decrease in the cells, even when protein synthesis was inhibited with chloramphenicol. In the presence of olive oil as a carbon source, nonionic detergents, such as Tween 80, increased extracellular lipase activity twofold. Like hexadecane, Tween 80 appeared to stimulate lipase secretion, although in a more disruptive manner, since other, normally nonsecreted proteins were found in the culture supernatant. Additionally, like olive oil, Tween 80 was found to induce lipase gene expression in strain PG1 in medium containing sucrose as a carbon source but not in glucose-containing medium, suggesting that lipase gene expression is prone to catabolite repression. In contrast, lipase production in the lipase-overproducing strain LU8093 was independent of the presence of an inducer and was not inhibited by glucose. In conclusion, hexadecane and Tween 80 enhance lipase production in
B. glumae
, and they act via different mechanisms.
A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid
Authors: Mruk Iwona , Kaczorowski Tadeusz
Published: April 2007

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from
Bacillus stearothermophilus
14P and Csp231I from
Citrobacter
sp. strain RFL231, both isospecific to the prototype HindIII R-M system.
Engineering Central Metabolic Pathways for High-Level Flavonoid Production in
Escherichia coli
Authors: Leonard Effendi , Lim Kok-Hong , Saw Phan-Nee , Koffas Mattheos A. G.
Published: April 2007

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in
Escherichia coli
were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from
Photorhabdus luminescens
(PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirA

Pl

) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with
Escherichia coli
BirA (BirA

Ec

) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirA

Ec

and the C terminus of BirA

Pl

. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.
Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton
Authors: Ivanikova Natalia V. , Popels Linda C. , McKay R. Michael L. , Bullerjahn George S.
Published: April 2007

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene andcpcBAPCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marineSynechococcusandProchlorococcus. Two new groups ofSynechococcusspp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwaterSynechococcusdiversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, thecpcBAlibrary from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of thecpcBAsequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105cells ml−1and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.
Repeat Domain Diversity of
avrBs3
-Like Genes in
Ralstonia solanacearum
Strains and Association with Host Preferences in the Field
Authors: Heuer Holger , Yin Yan-Ni , Xue Qing-Yun , Smalla Kornelia , Guo Jian-Hua
Published: April 2007

Genes homologous to
avrBs3
of
Xanthomonas
were detected in 309 strains of
Ralstonia solanacearum
biovars 3, 4, and 5 but not biovar 1 or 2. A statistically significant association between the originating plant species and internal repeats of the gene was found. Sequences of repeats and variation between nearly clonal strains revealed evidence of frequent recombination.
Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes
Authors: Mingardon Florence , Chanal Angélique , López-Contreras Ana M. , Dray Cyril , Bayer Edward A. , Fierobe Henri-Pierre
Published: April 2007

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from
Clostridium cellulolyticum
into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by
Neocallimastix patriciarum
, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from
C. cellulolyticum
. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from
N. patriciarum
bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.
Efficient Method of Cloning the Obligate Intracellular Bacterium
Coxiella burnetii
Authors: Beare Paul A. , Howe Dale , Cockrell Diane C. , Heinzen Robert A.
Published: April 2007

Coxiella burnetii
is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning
C. burnetii
are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual
C. burnetii
-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of
C. burnetii
. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining
C. burnetii
clones.
Relationship between Sublethal Injury and Inactivation of Yeast Cells by the Combination of Sorbic Acid and Pulsed Electric Fields
Authors: Somolinos M. , García D. , Condón S. , Mañas P. , Pagán R.
Published: April 2007

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts,Dekkera bruxellensisandSaccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of survivingD. bruxellensiscells and 99% of survivingS. cerevisiaecells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log105 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.
Aerobic Anoxygenic Phototrophic Bacteria Attached to Particles in Turbid Waters of the Delaware and Chesapeake Estuaries
Authors: Waidner Lisa A. , Kirchman David L.
Published: April 2007

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that, if abundant, may be biogeochemically important in the oceans. We used epifluorescence microscopy and quantitative PCR (qPCR) to examine the abundance of these bacteria by enumerating cells with bacteriochlorophyll
a
(bChl
a
) and the light-reaction center gene
pufM
, respectively. In the surface waters of the Delaware estuary, AAP bacteria were abundant, comprising up to 34% of prokaryotes, although the percentage varied greatly with location and season. On average, AAP bacteria made up 12% of the community as measured by microscopy and 17% by qPCR. In the surface waters of the Chesapeake, AAP bacteria were less abundant, averaging 6% of prokaryotes. AAP bacterial abundance was significantly correlated with light attenuation (
r
= 0.50) and ammonium (
r
= 0.42) and nitrate (
r
= 0.71) concentrations. Often, bChl
a
-containing bacteria were mostly attached to particles (31 to 94% of total AAP bacteria), while usually 20% or less of total prokaryotes were associated with particles. Of the cells containing
pufM
, up to 87% were associated with particles, but the overall average of particle-attached cells was 15%. These data suggest that AAP bacteria are particularly competitive in these two estuaries, in part due to attachment to particles.
Bacterial Diversity Associated with Blood Falls, a Subglacial Outflow from the Taylor Glacier, Antarctica
Authors: Mikucki Jill A. , Priscu John C.
Published: April 2007

Blood Falls is the surface manifestation of brine released from below the Taylor Glacier, McMurdo Dry Valleys, Antarctica. Geochemical analyses of Blood Falls show that this brine is of a marine origin. The discovery that 74% of clones and isolates from Blood Falls share high 16S rRNA gene sequence homology with phylotypes from marine systems supports this contention. The bacterial 16S rRNA gene clone library was dominated by a phylotype that had 99% sequence identity with
Thiomicrospira arctica
(46% of the library), a psychrophilic marine autotrophic sulfur oxidizer. The remainder of the library contained phylotypes related to the classes
Betaproteobacteria
,
Deltaproteobacteria
, and
Gammaproteobacteria
and the division
Bacteroidetes
and included clones whose closest cultured relatives metabolize iron and sulfur compounds. These findings are consistent with the high iron and sulfate concentrations detected in Blood Falls, which are likely due to the interactions of the subglacial brine with the underlying iron-rich bedrock. Our results, together with previous reports, suggest that the brine below the Taylor Glacier hosts a viable ecosystem with microorganisms capable of growth, supported by chemical energy present in reduced iron and sulfur compounds. The metabolic and phylogenetic structure of this subglacial microbial assemblage appears to be controlled by glacier hydrology, bedrock lithology, and the preglacial ecosystem.
Multilocus Sequence Typing Confirms the Close Genetic Interrelatedness of Three Distinct Flavescence Dorée Phytoplasma Strain Clusters and Group 16SrV Phytoplasmas Infecting Grapevine and Alder in Europe
Authors: Arnaud Guillaume , Malembic-Maher Sylvie , Salar Pascal , Bonnet Patrick , Maixner Michael , Marcone Carmine , Boudon-Padieu Elisabeth , Foissac Xavier
Published: April 2007

Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin
Scaphoideus titanus
. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci
map
and
uvrB
-
degV
, along with the sequence of the
secY
gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.
Competitive Fitness of Isolates Enriched on Phenanthrene Sorbed to Model Phases
Authors: Colores Gregory M. , Ward David M. , Inskeep William P.
Published: April 2007

We evaluated the competitive fitness of three phenanthrene-mineralizing isolates that were provided with nonsorbed or sorbed phenanthrene in competition experiments. One isolate had a clear advantage when presented with Amberlite IRC-50-sorbed phenanthrene, yet none were favored when presented with nonsorbed or Amberlite XAD-7-sorbed phenanthrene. Our results indicate that contaminant availability alone does not determine the isolates’ competitive fitness.
Alteration of Soil Rhizosphere Communities following Genetic Transformation of White Spruce
Authors: LeBlanc Philippe M. , Hamelin Richard C. , Filion Martin
Published: April 2007

The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through
Bacillus thuringiensis
transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first
B. thuringiensis
-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (
Picea glauca
) genetically modified by biolistic insertion of the
cryIA
(
b
),
uidA
(beta-glucuronidase), and
nptII
genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes
uidA
and
nptII
have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (
P
< 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the
cryIA
(
b
),
uidA
, and
nptII
transgenes, trees carrying only the
uidA
and
nptII
transgenes, and control trees. Clear rhizosphere microbial community alterations due to
B. thuringiensis
tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to
B. thuringiensis
genetic transformation and also to the impact of commonly used
uidA
and
nptII
genetic marker genes.
Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains
Authors: Guillén D. , Santiago M. , Linares L. , Pérez R. , Morlon J. , Ruiz B. , Sánchez S. , Rodríguez-Sanoja R.
Published: April 2007

The
Lactobacillus amylovorus
alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.
Presence and Sources of Fecal Coliform Bacteria in Epilithic Periphyton Communities of Lake Superior
Authors: Ksoll Winfried B. , Ishii Satoshi , Sadowsky Michael J. , Hicks Randall E.
Published: April 2007

Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and
Escherichia coli
were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 10
5
CFU cm
−2
by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the
E. coli
bacteria isolated from these periphyton communities could be identified when compared with a library of
E. coli
fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton
E. coli
strains that could be identified. Several periphyton
E. coli
isolates were genotypically identical (≥92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified
E. coli
strains from periphyton were added to the known source library, then 57% to 81% of
E. coli
strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated
E. coli
rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that
E. coli
from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.
A Novel
Acidimicrobium
Species in Continuous Cultures of Moderately Thermophilic, Mineral-Sulfide-Oxidizing Acidophiles
Authors: Cleaver Adam A. , Burton Nicolas P. , Norris Paul R.
Published: April 2007

A novel species of
Acidimicrobium
appeared to be the predominant ferrous iron oxidizer in a mixed culture that effected the continuous, efficient extraction of nickel from a mineral concentrate at 49°C, but it was not isolated in pure culture. It outcompeted
Acidimicrobium ferrooxidans
, which was expected to have a major role in iron oxidation in reactors gassed with air, and was outnumbered at 49°C only by the sulfur-oxidizing
Acidithiobacillus caldus. Sulfobacillus
species were expected to compete with
Acidimicrobium
species when culture aeration was enriched with carbon dioxide, but they were a minor component of the populations with and without this enrichment.
Sulfobacillus thermosulfidooxidans
replaced the
Acidimicrobium
species and
Acidithiobacillus caldus
when the temperature was increased to 55°C.
Thermoregulation of
N
-Acyl Homoserine Lactone-Based Quorum Sensing in the Soft Rot Bacterium
Pectobacterium atrosepticum
Authors: Latour Xavier , Diallo Stéphanie , Chevalier Sylvie , Morin Danièle , Smadja Bruno , Burini Jean-François , Haras Dominique , Orange Nicole
Published: April 2007

The psychrotolerant bacterium
Pectobacterium atrosepticum
produces four
N
-acyl homoserine lactones under a wide range of temperatures. Their thermoregulation differs from that of the exoenzyme production, described as being under quorum-sensing control. A mechanism involved in this thermoregulation consists of controlling
N
-acyl homoserine lactones synthase production at a transcriptional level.
Multivariate Logistic Regression for Predicting Total Culturable Virus Presence at the Intake of a Potable-Water Treatment Plant: Novel Application of the Atypical Coliform/Total Coliform Ratio
Authors: Black L. E. , Brion G. M. , Freitas S. J.
Published: April 2007

Predicting the presence of enteric viruses in surface waters is a complex modeling problem. Multiple water quality parameters that indicate the presence of human fecal material, the load of fecal material, and the amount of time fecal material has been in the environment are needed. This paper presents the results of a multiyear study of raw-water quality at the inlet of a potable-water plant that related 17 physical, chemical, and biological indices to the presence of enteric viruses as indicated by cytopathic changes in cell cultures. It was found that several simple, multivariate logistic regression models that could reliably identify observations of the presence or absence of total culturable virus could be fitted. The best models developed combined a fecal age indicator (the atypical coliform [AC]/total coliform [TC] ratio), the detectable presence of a human-associated sterol (epicoprostanol) to indicate the fecal source, and one of several fecal load indicators (the levels of
Giardia
species cysts, coliform bacteria, and coprostanol). The best fit to the data was found when the AC/TC ratio, the presence of epicoprostanol, and the density of fecal coliform bacteria were input into a simple, multivariate logistic regression equation, resulting in 84.5% and 78.6% accuracies for the identification of the presence and absence of total culturable virus, respectively. The AC/TC ratio was the most influential input variable in all of the models generated, but producing the best prediction required additional input related to the fecal source and the fecal load. The potential for replacing microbial indicators of fecal load with levels of coprostanol was proposed and evaluated by multivariate logistic regression modeling for the presence and absence of virus.
Salinity Constraints on Subsurface Archaeal Diversity and Methanogenesis in Sedimentary Rock Rich in Organic Matter
Authors: Waldron Patricia J. , Petsch Steven T. , Martini Anna M. , Nüsslein Klaus
Published: April 2007

The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl
−
in a subsurface shale rich in CH
4
derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH
4
-producing zone lacked electron acceptors such as O
2
, NO
3
−
, Fe
3+
, or SO
4
2−
. Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl
−
concentrations above ∼1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H
2
/CO
2
) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic
Methanohalophilus
species and a hydrogenotrophic
Methanoplanus
species at high salinity and a single phylotype closely related to
Methanocorpusculum bavaricum
at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH
4
-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.
Stable Isotope Ratios and Forensic Analysis of Microorganisms
Authors: Kreuzer-Martin Helen W. , Jarman Kristin H.
Published: April 2007

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures ofBacillus subtilis6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.
Biodegradation Processes in a Laboratory-Scale Groundwater Contaminant Plume Assessed by Fluorescence Imaging and Microbial Analysis
Authors: Rees Helen C. , Oswald Sascha E. , Banwart Steven A. , Pickup Roger W. , Lerner David N.
Published: April 2007

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O
2
(aq)] concentration field quantified the depletion of O
2
(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O
2
(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.
Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas
Authors: Willi Barbara , Boretti Felicitas S. , Meli Marina L. , Bernasconi Marco V. , Casati Simona , Hegglin Daniel , Puorger Maria , Neimark Harold , Cattori Valentino , Wengi Nicole , Reusch Claudia E. , Lutz Hans , Hofmann-Lehmann Regina
Published: April 2007

Three hemotropic mycoplasmas have been identified in pet cats:Mycoplasma haemofelis, “CandidatusMycoplasma haemominutum,” and “CandidatusMycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a fewIxodessp. andRhipicephalussp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequentlyMycoplasma coccoidesPCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection.M. haemofelisand “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.
Flux Analysis of Central Metabolic Pathways in
Geobacter metallireducens
during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid
Authors: Tang Yinjie J. , Chakraborty Romy , Martín Héctor García , Chu Jeannie , Hazen Terry C. , Keasling Jay D.
Published: April 2007

We analyzed the carbon fluxes in the central metabolism of
Geobacter metallireducens
strain GS-15 using
13
C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that
G. metallireducens
contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO
2
via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in
G. metallireducens
.
Using the
Ralstonia solanacearum
Tat Secretome To Identify Bacterial Wilt Virulence Factors
Authors: González Enid T. , Brown Darby G. , Swanson Jill K. , Allen Caitilyn
Published: April 2007

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen
Ralstonia solanacearum
, we mutated
tatC
, a key component of the
t
win-
a
rginine
t
ranslocation (Tat) secretion system. The
R. solanacearum tatC
mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the
R. solanacearum
strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The
R. solanacearum tatC
strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to
R. solanacearum
virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.
Sensitivity of
Escherichia albertii
, a Potential Food-Borne Pathogen, to Food Preservation Treatments
Authors: Sharma Manan , Kniel Kalmia E. , Derevianko Alexandra , Ling Jason , Bhagwat Arvind A.
Published: April 2007

Escherichia albertii
is a potential food-borne pathogen because of its documented ability to cause diarrheal disease by producing attachment and effacement lesions. Its tolerances to heat (56°C), acid (pH 3.0), and pressure (500 MPa [5 min]) were evaluated and found to be significantly less than those of wild-type
E. coli
O157:H7.
Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote
Entamoeba histolytica
Authors: Davis Carrie A. , Brown Michael P. S. , Singh Upinder
Published: April 2007

Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In
Entamoeba histolytica
, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the
E. histolytica
U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in
E. histolytica
gene expression.
Molecular Basis for Preferential Protective Efficacy of Antibodies Directed to the Poorly Acetylated Form of Staphylococcal Poly-
N
-Acetyl-β-(1-6)-Glucosamine
Authors: Cerca Nuno , Jefferson Kimberly K. , Maira-Litrán Tomas , Pier Danielle B. , Kelly-Quintos Casie , Goldmann Donald A. , Azeredo Joana , Pier Gerald B.
Published: May 2007

Poly-
N
-acetyl-glucosamine (PNAG) is a staphylococcal surface polysaccharide influencing biofilm formation that is also under investigation for its vaccine potential. Antibodies that bind to PNAG with either low (<15%) or high (>90%) levels of acetate are superior at opsonic and protective activity compared with antibodies that bind to PNAG with only high levels (>70%) of acetate. PNAG is synthesized by four proteins encoded within the intercellular adhesin (
ica
) locus
icaADBC
. In
Staphylococcus epidermidis
,
icaB
encodes a deacetylase needed for the surface retention of PNAG and optimal biofilm formation. In this study, we confirmed that
icaB
plays a similar role in
Staphylococcus aureus
and found that an
icaB
mutant of
S. aureus
expressed significantly less surface-associated PNAG, was highly susceptible to antibody-independent opsonic killing that could not be enhanced with antibody raised against deacetylated PNAG (dPNAG), and had reduced survival capacity in a murine model of bacteremia. In contrast, an
icaB
-overexpressing strain produced primarily surface-associated PNAG, was more susceptible to opsonophagocytosis with antibody to dPNAG, and had increased survival in a murine bacteremia model. The highly acetylated secreted PNAG was more effective at blocking opsonic killing mediated by a human monoclonal antibody (mAb) to native PNAG than it was at blocking killing mediated by a human mAb to dPNAG, which by itself was a more effective opsonin. Retention of dPNAG on the surface of
S. aureus
is key to increased survival during bacteremia and also provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG.