bluuebunny/crossref_metadata_2025 huggingface dataset:
σB-Dependent and σB-Independent Mechanisms Contribute to Transcription ofListeria monocytogenesCold Stress Genes during Cold Shock and Cold Growth
Authors: Chan Yvonne C. , Boor Kathryn J. , Wiedmann Martin
Published: August 2007

The role of the stress response regulator σB(encoded bysigB) in directing the expression of selected putative and confirmed cold response genes was evaluated usingListeria monocytogenes10403S and an isogenic ΔsigBmutant, which were either cold shocked at 4°C in brain heart infusion (BHI) broth for up to 30 min or grown at 4°C in BHI for 12 days. Transcript levels of the housekeeping genesrpoBandgap, the σB-dependent genesopuCAandbsh, and the cold stress genesltrC, oppA, andfriwere measured using quantitative reverse transcriptase PCR. Transcriptional start sites forltrC, oppA, andfriwere determined using rapid amplification of cDNA ends PCR. Centrifugation was found to rapidly induce σB-dependent transcription, which necessitated the use of centrifugation-independent protocols to evaluate the contributions of σBto transcription during cold shock. Our data confirmed that transcription of the cold stress genesltrCandfriis at least partially σBdependent and experimentally identified a σB-dependentltrCpromoter. In addition, our data indicate that (i) while σBactivity is induced during 30 min of cold shock, this cold shock does not induce the transcription of σB-dependent or -independent cold shock genes; (ii) σBis not required forL. monocytogenesgrowth at 4°C in BHI; and (iii) transcription of the putative cold stress genesopuCA, fri, andoppAis σBindependent during growth at 4°C, while bothbshandltrCshow growth phase and σB-dependent transcription during growth at 4°C. We conclude that σB-dependent and σB-independent mechanisms contribute to the ability ofL. monocytogenesto survive and grow at low temperatures.
The Motility Symbiont of the Termite Gut FlagellateCaduceia versatilisIs a Member of the “Synergistes” Group
Authors: Hongoh Yuichi , Sato Tomoyuki , Dolan Michael F. , Noda Satoko , Ui Sadaharu , Kudo Toshiaki , Ohkuma Moriya
Published: August 2007

The flagellateCaduceia versatilisin the gut of the termiteCryptotermes cavifronsreportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with theCaduceiacells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the orderBacteroidalesin theBacteroidetesphylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; theBacteroidalesphylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “CandidatusTammella caduceiae,” for this unique motility ectosymbiont ofCaducaia versatilis.
Isolation ofSalmonella entericaSerovar Enteritidis from Houseflies (Musca domestica) Found in Rooms ContainingSalmonellaSerovar Enteritidis-Challenged Hens
Authors: Holt Peter S. , Geden Christopher J. , Moore Randle W. , Gast Richard K.
Published: August 2007

Houseflies (Musca domestica) released into rooms containing hens challenged withSalmonella entericaserovar Enteritidis (Salmonellaserovar Enteritidis) rapidly became contaminated withSalmonellaserovar Enteritidis. Forty to 50% of the flies were contaminated at 48 h, and the percentage increased to 50 to 70% at 4 and 7 days postexposure and then decreased to 30% at day 15. Initial attempts at recovering surface organisms for culture using an aqueous rinse were largely unsuccessful, while cultures of internal contents readily recoveredSalmonellaserovar Enteritidis. However, when 0.5% detergent was incorporated into the rinse, high recovery levels of bacteria were observed from both external and internal culture regimens, indicating equal distribution of the organism on and in the fly and a tighter interaction of the organism with the host than previously thought.Salmonellaserovar Enteritidis was isolated routinely from the fly gut, on rare occasions from the crop, and never from the salivary gland. Feeding contaminated flies to hens resulted in gut colonization of a third of the birds, but release of contaminated flies in a room containing previously unchallenged hens failed to result in colonization of any of the subject birds. These results indicate that flies exposed to an environment containingSalmonellaserovar Enteritidis can become colonized with the organism and might serve as a source for transmission ofSalmonellaserovar Enteritidis within a flock situation.
Survival of
Mycobacterium avium
in Drinking Water Biofilms as Affected by Water Flow Velocity, Availability of Phosphorus, and Temperature
Authors: Torvinen Eila , Lehtola Markku J. , Martikainen Pertti J. , Miettinen Ilkka T.
Published: August 2007

Mycobacterium avium
is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of
M. avium
in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4′,6′-diamidino-2-phenylindole) staining, and concentrations of
M. avium
were determined by culture and fluorescence in situ hybridization methods. The spiked
M. avium
survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 μg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of
M. avium
. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of
M. avium
, although it increased the growth of biofilms. A higher temperature (20°C versus 7°C) increased both the number of heterotrophic bacteria and the survival of
M. avium
in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing
M. avium
in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.
Biofilm Interactions between Distinct Bacterial Genera Isolated from Drinking Water
Authors: Simões Lúcia Chaves , Simões Manuel , Vieira Maria João
Published: August 2007

In the environment, multiple microorganisms coexist as communities, competing for resources and often associated as biofilms. In this study, single- and dual-species biofilm formation by, and specific activities of, six heterotrophic intergeneric bacteria were determined using 96-well polystyrene plates over a 72-h period. These bacteria were isolated from drinking water and identified by partial 16S rRNA gene sequencing. A series of planktonic studies was also performed, assessing the bacterial growth rate, motility, and production of quorum-sensing inhibitors (QSI). This constituted an attempt to identify key attributes allowing bacteria to effectively interact and coexist in a drinking-water environment. We observed that in both pure and dual cultures, all of the isolates formed stable biofilms within 72 h, with specific metabolic activity decreasing, in most cases, with an increase in biofilm mass. The largest single- and dual-biofilm amounts were found for
Methylobacterium
sp. and the combination of
Methylobacterium
sp. and
Mycobacterium mucogenicum
, respectively. Evidences of microbial interactions in dual-biofilm formation, associated with appreciable biomass variation in comparison with single biofilms, were found for the following cases: synergy/cooperation between
Sphingomonas capsulata
and
Burkholderia cepacia, S. capsulata
and
Staphylococcus
sp., and
B. cepacia
and
Acinetobacter calcoaceticus
and antagonism between
S. capsulata
and
M. mucogenicum, S. capsulata
and
A. calcoaceticus
, and
M. mucogenicum
and
Staphylococcus
sp. A neutral interaction was found for
Methylobacterium
sp.-
M. mucogenicum, S. capsulata
-
Staphylococcus
sp.,
M. mucogenicum
-
A. calcoaceticus
, and
Methylobacterium
sp.-
A. calcoaceticus
biofilms, since the resultant dual biofilms had a mass and specific metabolic activity similar to the average for each single biofilm.
B. cepacia
had the highest growth rate and motility and produced QSI. Other bacteria producing QSI were
Methylobacterium
sp.,
S. capsulata
, and
Staphylococcus
sp. However, only for
S. capsulata
-
M. mucogenicum, S. capsulata
-
A. calcoaceticus
, and
M. mucogenicum
-
Staphylococcus
sp., dual-biofilm formation seems to be regulated by the QSI produced by
S. capsulata
and
Staphylococcus
sp. and by the increased growth rate of
S. capsulata
. The parameters assessed by planktonic studies did not allow prediction and generalization of the exact mechanism regulating dual-species biofilm formation between the drinking-water bacteria.
Optimization of Airborne Endotoxin Exposure Assessment: Effects of Filter Type, Transport Conditions, Extraction Solutions, and Storage of Samples and Extracts
Authors: Spaan Suzanne , Heederik Dick J. J. , Thorne Peter S. , Wouters Inge M.
Published: August 2007

Endotoxin exposure occurs in homes and occupational environments and is known to cause adverse health effects. In order to compare results from different studies and establish standards, airborne endotoxin exposures should be assessed using standardized methods. Although the European Committee for Standardization (CEN) developed guidelines for endotoxin exposure assessment, these leave room for individual interpretation. The influence of methods of sampling, extraction, and analysis has never been investigated in a full experimental design. Thus, we sought to fully elucidate the importance of all facets of endotoxin assessment. Inhalable dust samples collected simultaneously were used to investigate the effects on and interactions with airborne endotoxin concentration in two working environments of filter type (glass fiber or Teflon), transport conditions (with/without desiccant), sample storage (−20 or 4°C), extraction solution (pyrogen-free water [PFW] or PFW plus 0.05% Tween 20), extract storage (−20 or 4°C), and assay solution (PFW or PFW plus 0.05% Tween 20). Four hundred samples were collected and randomly distributed over the 20 combinations of treatments. There were no differences found for transport conditions and storage temperature of extracts. Also, no interactions between study variables existed. Sampling on glass-fiber filters, storage of samples in the freezer, and extraction in PFW plus 0.05% Tween 20 resulted in 1.3-, 1.1-, and 2.1-fold-higher estimated endotoxin concentrations, respectively. Use of PFW plus 0.05% Tween 20 in the assay solution had an additive effect. Thus, this study investigated gaps in the CEN protocol and provides data with which to fully specify a protocol for standardization of endotoxin exposure assessment.
Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs
Authors: Scheirlinck Ilse , Van der Meulen Roel , Van Schoor Ann , Vancanneyt Marc , De Vuyst Luc , Vandamme Peter , Huys Geert
Published: August 2007

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the generaLactobacillus, Pediococcus, Leuconostoc, Weissella, andEnterococcus, with the heterofermentative speciesLactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, andLactobacillus pontisas the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
Nitrogen Fixation by
Vibrio parahaemolyticus
and Its Implications for a New Ecological Niche
Authors: Criminger J. D. , Hazen T. H. , Sobecky P. A. , Lovell C. R.
Published: August 2007

A
Vibrio parahaemolyticus
strain isolated from the rhizosphere of the ecosystem dominant estuarine grass,
Spartina alterniflora
, was characterized and shown to carry
nifH
, the gene encoding the nitrogenase iron protein, and to fix N
2
. Nitrogen fixation may contribute substantially to the adaptability, niche breadth, and ecological significance of
V. parahaemolyticus
.
Novel Sensitive High-Throughput Screening Strategy for Nitrilase-Producing Strains
Authors: Zhu Qing , Fan Ao , Wang Yuanshan , Zhu Xiaoqin , Wang Zhao , Wu Minghuo , Zheng Yuguo
Published: August 2007

Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe:
o
-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases,
o
-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb
3+
, serve as a photon antenna and sensitize Tb
3+
luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.
IncP-1β Plasmid pGNB1 Isolated from a Bacterial Community from a Wastewater Treatment Plant Mediates Decolorization of Triphenylmethane Dyes
Authors: Schlüter Andreas , Krahn Irene , Kollin Florian , Bönemann Gabriele , Stiens Michael , Szczepanowski Rafael , Schneiker Susanne , Pühler Alfred
Published: August 2007

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1β plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene
trfA
and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn
5501
and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene
tmr
and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS
1071
and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1
tmr
gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the α-proteobacterium
Sinorhizobium meliloti
and the γ-proteobacterium
Escherichia coli
. This is the first report of a promiscuous IncP-1β plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.
“
Candidatus
Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes
Authors: He Shaomei , Gall Daniel L. , McMahon Katherine D.
Published: August 2007

We investigated the fine-scale population structure of the “
Candidatus
Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (
ppk1
) as a genetic marker. We retrieved fragments of “
Candidatus
Accumulibacter” 16S rRNA and
ppk1
genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and
ppk1
were largely congruent, with
ppk1
granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “
Candidatus
Accumulibacter” lineage. Sequences from at least five clades of “
Candidatus
Accumulibacter” were recovered by
ppk1
-targeted PCR, and subsequently, specific primer sets were designed to target the
ppk1
gene for each clade. Quantitative real-time PCR (qPCR) assays using “
Candidatus
Accumulibacter”-specific 16S rRNA and “
Candidatus
Accumulibacter” clade-specific
ppk1
primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “
Candidatus
Accumulibacter” lineage and the relative distributions and abundances of the five “
Candidatus
Accumulibacter” clades. The qPCR-based estimation of the total “
Candidatus
Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using
ppk1
, demonstrating the power of
ppk1
as a genetic marker for detection of all currently defined “
Candidatus
Accumulibacter” clades. The relative distributions of “
Candidatus
Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “
Candidatus
Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.
Bacteria Metabolically Engineered for Enhanced Phytochelatin Production and Cadmium Accumulation
Authors: Kang Seung Hyun , Singh Shailendra , Kim Jae-Young , Lee Wonkyu , Mulchandani Ashok , Chen Wilfred
Published: August 2007

Phytochelatins (PCs) with good binding affinities for a wide range of heavy metals were exploited to develop microbial sorbents for cadmium removal. PC synthase from
Schizosaccharomyces pombe
(SpPCS) was overexpressed in
Escherichia coli
, resulting in PC synthesis and 7.5-times-higher Cd accumulation. The coexpression of a variant γ-glutamylcysteine synthetase desensitized to feedback inhibition (GshI*) increased the supply of the PC precursor glutathione, resulting in further increases of 10- and 2-fold in PC production and Cd accumulation, respectively. A Cd transporter, MntA, was expressed with SpPCS and GshI* to improve Cd uptake, resulting in a further 1.5-fold increase in Cd accumulation. The level of Cd accumulation in this recombinant
E. coli
strain (31.6 μmol/g [dry weight] of cells) was more than 25-fold higher than that in the control strain.
An Sfi1p-Like Centrin-Binding Protein Mediates Centrin-Based Ca
2+
-Dependent Contractility in
Paramecium tetraurelia
Authors: Gogendeau Delphine , Beisson Janine , de Loubresse Nicole Garreau , Le Caer Jean-Pierre , Ruiz Françoise , Cohen Jean , Sperling Linda , Koll France , Klotz Catherine
Published: August 2007

The previous characterization and structural analyses of Sfi1p, a
Saccharomyces cerevisiae
centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca
2+
-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using
Paramecium tetraurelia
, which harbors a large Ca
2+
-dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the
Paramecium
genome sequence, we characterized the Pt
CenBP1
gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX
11
F/LX
2
WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant Pt
CenBP1
allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca
2+
-dependent contractile processes.
Plasmids pMOL28 and pMOL30 of
Cupriavidus metallidurans
Are Specialized in the Maximal Viable Response to Heavy Metals
Authors: Monchy Sébastien , Benotmane Mohammed A. , Janssen Paul , Vallaeys Tatiana , Taghavi Safiyh , van der Lelie Daniel , Mergeay Max
Published: August 2007

We fully annotated two large plasmids, pMOL28 (164 open reading frames [ORFs]; 171,459 bp) and pMOL30 (247 ORFs; 233,720 bp), in the genome of
Cupriavidus metallidurans
CH34. pMOL28 contains a backbone of maintenance and transfer genes resembling those found in plasmid pSym of
C. taiwanensis
and plasmid pHG1 of
C. eutrophus
, suggesting that they belong to a new class of plasmids. Genes involved in resistance to the heavy metals Co(II), Cr(VI), Hg(II), and Ni(II) are concentrated in a 34-kb region on pMOL28, and genes involved in resistance to Ag(I), Cd(II), Co(II), Cu(II), Hg(II), Pb(II), and Zn(II) occur in a 132-kb region on pMOL30. We identified three putative genomic islands containing metal resistance operons flanked by mobile genetic elements, one on pMOL28 and two on pMOL30. Transcriptomic analysis using quantitative PCR and microarrays revealed metal-mediated up-regulation of 83 genes on pMOL28 and 143 genes on pMOL30 that coded for all known heavy metal resistance proteins, some new heavy metal resistance proteins (
czcJ
,
mmrQ
, and
pbrU
), membrane proteins, truncated transposases, conjugative transfer proteins, and many unknown proteins. Five genes on each plasmid were down-regulated; for one of them,
chrI
localized on pMOL28, the down-regulation occurred in the presence of five cations. We observed multiple cross-responses (induction of specific metal resistance by other metals), suggesting that the cellular defense of
C. metallidurans
against heavy metal stress involves various regulons and probably has multiple stages, including a more general response and a more metal-specific response.
Control of Methionine Synthesis and Uptake by MetR and Homocysteine in
Streptococcus mutans
Authors: Sperandio Brice , Gautier Céline , McGovern Stephen , Ehrlich Dusko S. , Renault Pierre , Martin-Verstraete Isabelle , Guédon Eric
Published: August 2007

MetR (formerly Smu.1225), a regulator of the LysR family, controls key genes for methionine supply in
Streptococcus mutans
. An
S. mutans metR
mutant is unable to transport
l
-methionine and to grow in the absence of this amino acid. Accordingly, MetR activates transcription by binding to the promoter regions of two gene clusters and smu.1487, whose products are involved in methionine biosynthesis (MetEF and Smu.1487) and uptake (AtmBDE). Transcriptional activation by MetR requires the presence of a 17-bp palindromic sequence, the Met box. Base substitutions in the Met box hinder the formation of a MetR-DNA complex and abolish MetR-dependent activation, showing that Met boxes correspond to MetR recognition sites. Activation by MetR occurs in methionine-depleted medium and is rapidly triggered under nonactivating conditions by the addition of homocysteine. This intermediate of methionine biosynthesis increases the affinity of MetR for DNA in vitro and appears to be the MetR coeffector in vivo. Homocysteine plays a crucial role in methionine metabolic gene regulation by controlling MetR activity. A similar mechanism of homocysteine- and MetR-dependent control of methionine biosynthetic genes operates in
S. thermophilus
. These data suggest a common mechanism for the regulation of the methionine supply in streptococci. However, some streptococcal species are unable to synthesize the homocysteine coeffector. This intriguing feature is discussed in the light of comparative genomics and streptococcal ecology.
Characterization of IsaA and SceD, Two Putative Lytic Transglycosylases of
Staphylococcus aureus
Authors: Stapleton Melanie R. , Horsburgh Malcolm J. , Hayhurst Emma J. , Wright Lynda , Jonsson Ing-Marie , Tarkowski Andrej , Kokai-Kun John F. , Mond James J. , Foster Simon J.
Published: August 2007

Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division.
Staphylococcus aureus
has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the
isaA
and
sceD
genes are partially mutually compensatory and that the production of SceD is upregulated in an
isaA
mutant. The expression of
sceD
is also greatly upregulated by the presence of NaCl. Several regulators of
isaA
and
sceD
expression were identified. Inactivation of
sceD
resulted in impaired cell separation, as shown by light microscopy, and “clumping” of bacterial cultures. An
isaA sceD
mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.
Correlation between Growth Rates, EIIA
Crr
Phosphorylation, and Intracellular Cyclic AMP Levels in
Escherichia coli
K-12
Authors: Bettenbrock Katja , Sauter Thomas , Jahreis Knut , Kremling Andreas , Lengeler Joseph W. , Gilles Ernst-Dieter
Published: August 2007

In
Escherichia coli
K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA
Crr
(EIIA
Crr
) (= EIIA
Glc
) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA
Crr
(EIIA
Crr
∼P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA
Crr
to EIIA
Crr
∼P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h
−1
and 0.7 h
−1
, corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.
ASerratia marcescensOxyR Homolog Mediates Surface Attachment and Biofilm Formation
Authors: Shanks Robert M. Q. , Stella Nicholas A. , Kalivoda Eric J. , Doe Megan R. , O'Dee Dawn M. , Lathrop Kira L. , Guo Feng Li , Nau Gerard J.
Published: August 2007

OxyR is a conserved bacterial transcription factor with a regulatory role in oxidative stress response. From a genetic screen for genes that modulate biofilm formation in the opportunistic pathogenSerratia marcescens, mutations in anoxyRhomolog and predicted fimbria structural genes were identified.S. marcescens oxyRmutants were severely impaired in biofilm formation, in contrast to the hyperbiofilm phenotype exhibited byoxyRmutants ofEscherichia coliandBurkholderia pseudomallei. Further analysis revealed that OxyR plays a role in the primary attachment of cells to a surface. Similar to what is observed in other bacterial species,S. marcescensOxyR is required for oxidative stress resistance. Mutations inoxyRand type I fimbrial genes resulted in severe defects in fimbria-associated phenotypes, revealing roles in cell-cell and cell-biotic surface interactions. Transmission electron microscopy revealed the absence of fimbria-like surface structures on an OxyR-deficient strain and an enhanced fimbrial phenotype in strains bearingoxyRon a multicopy plasmid. The hyperfimbriated phenotype conferred by the multicopyoxyRplasmid was absent in a type I fimbrial mutant background. Real-time reverse transcriptase PCR indicated an absence of transcripts from a fimbrial operon in anoxyRmutant that were present in the wild type and a complementedoxyRmutant strain. Lastly, chromosomalPlac-mediated expression offimABCDwas sufficient to restore wild-type levels of yeast agglutination and biofilm formation to anoxyRmutant. Together, these data support a model in which OxyR contributes to early stages ofS. marcescensbiofilm formation by influencing fimbrial gene expression.
An Extremely Oligotrophic Bacterium,
Rhodococcus erythropolis
N9T-4, Isolated from Crude Oil
Authors: Ohhata Naoko , Yoshida Nobuyuki , Egami Hiroshi , Katsuragi Tohoru , Tani Yoshiki , Takagi Hiroshi
Published: August 2007

Rhodococcus erythropolis
N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO
2
-limiting conditions but could grow on a medium containing NaHCO
3
under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO
2
. Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol:
N
,
N
′-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from
Rhodococcus
sp. These enzyme activities were three times higher under oligotrophic conditions than under
n
-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO
2
fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of
R. erythropolis
N9T-4.
MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in
Sulfolobus solfataricus
Authors: Fiorentino Gabriella , Ronca Raffaele , Cannio Raffaele , Rossi Mosè , Bartolucci Simonetta
Published: August 2007

A DNA binding protein, BldR, was identified in the crenarchaeon
Sulfolobus solfataricus
as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the
bldR
gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the
mar
-like operon. The level of
mar
-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in
E. coli
, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and
mar
-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH
Ss
, stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.
The Archaeon
Methanosarcina acetivorans
Contains a Protein Disulfide Reductase with an Iron-Sulfur Cluster
Authors: Lessner Daniel J. , Ferry James G.
Published: August 2007

Methanosarcina acetivorans
, a strictly anaerobic methane-producing species belonging to the domain
Archaea
, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain
Bacteria
. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated. The product of MA3736, overproduced in
Escherichia coli
, had protein disulfide reductase activity dependent on a C
67
XXC
70
motif not found in carboxymuconolactone decarboxylase. We propose that MA3736 be renamed
mdrA
(
m
ethanosarcina
d
isulfide
r
eductase). Further, unlike carboxymuconolactone decarboxylase, MdrA contained an Fe-S cluster. Binding of the Fe-S cluster was dependent on essential cysteines C
67
and C
70
, while cysteines C
39
and C
107
were not required. Loss of the Fe-S cluster resulted in conversion of MdrA from an inactive hexamer to a trimer with protein disulfide reductase activity. The data suggest that MdrA is the prototype of a previously unrecognized protein disulfide reductase family which contains an intermolecular Fe-S cluster that controls oligomerization as a mechanism to regulate protein disulfide reductase activity.
CheX in the Three-Phosphatase System of Bacterial Chemotaxis
Authors: Muff Travis J. , Foster Richard M. , Liu Peter J. Y. , Ordal George W.
Published: August 2007

Bacterial chemotaxis involves the regulation of motility by a modified two-component signal transduction system. In
Escherichia coli
, CheZ is the phosphatase of the response regulator CheY but many other bacteria, including
Bacillus subtilis
, use members of the CheC-FliY-CheX family for this purpose. While
Bacillus subtilis
has only CheC and FliY, many systems also have CheX. The effect of this three-phosphatase system on chemotaxis has not been studied previously. CheX was shown to be a stronger CheY-P phosphatase than either CheC or FliY. In
Bacillus subtilis
, a
cheC
mutant strain was nearly complemented by heterologous
cheX
expression. CheX was shown to overcome the Δ
cheC
adaptational defect but also generally lowered the counterclockwise flagellar rotational bias. The effect on rotational bias suggests that CheX reduced the overall levels of CheY-P in the cell and did not truly replicate the adaptational effects of CheC. Thus, CheX is not functionally redundant to CheC and, as outlined in the discussion, may be more analogous to CheZ.
Functional Analysis of the
Mycoplasma genitalium
MG312 Protein Reveals a Specific Requirement of the MG312 N-Terminal Domain for Gliding Motility
Authors: Burgos Raul , Pich Oscar Q. , Querol Enrique , Piñol Jaume
Published: August 2007

The human pathogen
Mycoplasma genitalium
is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of
M. genitalium
cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the
M. genitalium
terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.
Mycobacterium mucogenicumfrom the Hickman line of an immunocompromised patient: Table 1
Authors: Marshall C , Samuel J , Galloway A , Pedler S
Published: August 2007

Gram stain of a positive blood culture is the clinician’s first indication of a possible causative infective organism and a guide to suitable antimicrobial therapy prior to cultural and phenotypic identification with susceptibility test results. Occasionally interpretation of a Gram stain can be difficult; if there is a low bacterial load, no organisms may be seen. Such a case is reported, where a positive blood culture taken from the Hickman line of an immunocompromised patient flagged as positive at 5 days’ incubation, but no organisms were seen on Gram film. On subculture, a slow growing Gram-positive bacillus was isolated which was initially misidentified and reported as a “diphtheroid” species. The actual identity of this organism and further isolates was later elucidated asMycobacterium mucogenicum, a rapidly growing non-tuberculous mycobacterium.
Traditional urinary cytology and tyrosinase RT-PCR in metastatic melanoma patients: correlation with clinical status
Authors: Savoia P , Osella-Abate S , Comessatti A , Nardò T , Marchiò C , Pacchioni D , Quaglino P , Bernengo M G
Published: August 2007

Background:

The finding of a suspicious urinary cytology is not uncommon in melanoma patients, in as much as morphology alone is often unable to distinguish the variable cytological features of melanoma cells. To date, although tyrosinase reverse transcription (RT)-PCR assay has been used to identify melanoma cells in peripheral blood and tissues, this method has not been applied to the analysis of urine samples.

Methods:

RT-PCR mRNA tyrosinase expression was analysed in 79 urine samples from patients with metastatic melanoma and correlated with standard morphology/immunocytology. The results were compared with the disease course and presence of genito-urinary involvement.

Results:

A positive RT-PCR expression was found in 18/79 urine samples from patients with metastases; four of the 18 patients had positive cytology, nine had atypical cytology, and five had negative cytology. Genito-urinary metastases were demonstrated in 27.8% tyrosinase-positive patients but in only 9.8% of the negative patients. The majority of tyrosinase-positive patients had a progressive disease unresponsive to chemotherapy. Urine samples from 20 patients with non-melanoma cancer and 20 healthy subjects were all negative.

Conclusions:

Our data demonstrate the higher sensitivity of RT-PCR compared with standard cytology in detection of urinary melanoma cells, and suggest that this assay could be used as an additional tool in the presence of negative or suspicious cytology.
Is microscopic examination of hysterectomy specimens removed for clinically benign disease necessary?
Authors: Andrews T , Monaghan H
Published: August 2007

The likelihood of finding significant pathology in a hysterectomy specimen undertaken for clinically benign disease is low. Some data indicate that microscopic examination of these specimens may be abandoned. While this approach may seem attractive, the authors believe that there are a number of practical and clinical governance issues associated with this. Instead a pragmatic approach is advocated, with two tissue blocks (cervix and endomyometrium) being adequate in most cases.
Effect of Lean method implementation in the histopathology section of an anatomical pathology laboratory
Authors: Raab S S , Grzybicki D M , Condel J L , Stewart W R , Turcsanyi B D , Mahood L K , Becich M J
Published: August 2007

Background:

In the USA, the lack of processes standardisation in histopathology laboratories leads to less than optimal quality, errors, inefficiency and increased costs. The effectiveness of large-scale quality improvement initiatives has been evaluated rarely.

Aim:

To measure the effect of implementation of a Lean quality improvement process on the efficiency and quality of a histopathology laboratory section.

Methods:

A non-concurrent interventional cohort study from 1 January 2003 to 31 December 2006 was performed, and the Lean process was implemented on 1 January 2004. Also compared was the productivity of the Lean histopathology section to a sister histopathology section that did not implement Lean processes. Pre- and post-Lean specimen turnaround time and productivity ratios (work units/full time equivalents) were measured. For 200 Lean interventions, a 5-part Likert scale was used to assess the impact on error, success and complexity.

Results:

In the Lean laboratory, the mean monthly productivity ratio increased from 3439 to 4074 work units/full time equivalents (p<0.001) as the mean daily histopathology section specimen turnaround time decreased from 9.7 to 9.0 h (p = 0.01). The Lean histopathology section had a higher productivity ratio compared with a sister histopathology section (1598 work units/full time equivalents, p<0.001) that did not implement Lean processes. The mean impact, success and complexity of interventions were 2.4, 2.7 and 2.5, respectively. The mean number of specific error causes affected by individual interventions was 2.6.

Conclusion:

It is concluded that Lean process implementation improved efficiency and quality in the histopathology section.
A systematic review of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis
Authors: Steingart Karen R , Henry Megan , Laal Suman , Hopewell Philip C , Ramsay Andrew , Menzies Dick , Cunningham Jane , Weldingh Karin , Pai Madhukar
Published: August 2007

Conventional diagnostic tests for tuberculosis have several limitations and are often unhelpful in establishing the diagnosis of extrapulmonary tuberculosis. Although commercial serological antibody based tests are available, their usefulness in the diagnosis of extrapulmonary tuberculosis is unknown. A systematic review was conducted to assess the accuracy of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis. In a comprehensive search, 21 studies that reported data on sensitivity and specificity for extrapulmonary tuberculosis were identified. These studies evaluated seven different commercial tests, with Anda-TB IgG accounting for 48% of the studies. The results showed that (1) all commercial tests provided highly variable estimates of sensitivity (range 0.00–1.00) and specificity range 0.59–1.00 for all extrapulmonary sites combined; (2) the Anda-TB IgG kit showed highly variable sensitivity (range 0.26–1.00) and specificity (range 0.59–1.00) for all extrapulmonary sites combined; (3) for all tests combined, sensitivity estimates for both lymph node tuberculosis (range 0.23–1.00) and pleural tuberculosis (range 0.26–0.59) were poor and inconsistent; and (4) there were no data to determine the accuracy of the tests in children or in patients with HIV infection, the two groups for which the test would be most useful. At present, commercial antibody detection tests for extrapulmonary tuberculosis have no role in clinical care or case detection.
REGION-BASED AND CONTENT ADAPTIVE SKIN DETECTION IN COLOR IMAGES
Authors: CHEN WEI-CHE , WANG MING-SHI
Published: August 2007

Skin detection plays an important role in applications such as face detection and tracking, person detection and pornography detection. While previous studies focus on pixel-based skin color detection techniques that individually classify each pixel as skin color or non-skin color, this study presents a region-based algorithm for detecting skin color. The proposed algorithm uses a special region, called key skin region, as the basis to classify skin color. A performance comparison with conventional skin classifiers, including the Bayesian classifier, the unimodal Gaussian classifier and the Gaussian mixture classifier, is made in this study. Experimental results show that the proposed algorithm outperforms other tested skin classifiers. Furthermore, the skin regions detected by the proposed algorithm, especially facial regions, are nearly complete with no hollow holes in these regions. This property can simplify the complexity of implementing applications that use skin color as their basis, such as face detection and face tracking.