bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Pattern of α
s1
- and β-Casein Breakdown and Release of a Bioactive Peptide by a Cell Envelope Proteinase from
Lactobacillus delbrueckii
subsp.
lactis
CRL 581
Authors: Hebert Elvira María , Mamone Gianfranco , Picariello Gianluca , Raya Raúl R. , Savoy Graciela , Ferranti Pasquale , Addeo Francesco
Published: April 2008

The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter
Lactobacillus delbrueckii
subsp.
lactis
CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of
L. delbrueckii
subsp.
lactis
CRL 581 exhibited a mixed-type CEP
I/III
variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the α
s1
- and β-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of α
s1
- and β-casein breakdown was defined and is reported herein, this being the first report for a CEP of
L. delbrueckii
subsp.
lactis
. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.
Longitudinal Study of the Molecular Epidemiology of
Campylobacter jejuni
in Cattle on Dairy Farms
Authors: Kwan Patrick S. L. , Birtles Andrew , Bolton Frederick J. , French Nigel P. , Robinson Susan E. , Newbold Lynne S. , Upton Mathew , Fox Andrew J.
Published: April 2008

Multilocus sequence typing (MLST), an accurate and phylogenetically robust characterization method for population studies of
Campylobacter
, was applied to
Campylobacter jejuni
isolates (
n
= 297) from the fecal samples of cattle from five dairy farms in Cheshire, United Kingdom, collected throughout 2003. The population dynamics of the
C. jejuni
strains, as identified by the occurrence of sequence types and clonal complexes, demonstrated variations within and between cattle populations over time. Three clonal lineages have emerged to predominate among the cattle isolates, namely, the ST-61 complex (24.2%), ST-21 complex (23.6%), and ST-42 complex (20.5%). This provided further evidence that the ST-61 clonal complex may present a cattle-adapted
C. jejuni
genotype. In addition, the ST-42 clonal complex may also represent an important cattle-associated genotype. Strong geographical associations for these genotypes were also found among the farms. This is the first longitudinal study and the largest study to date for
C. jejuni
involving cattle populations using MLST for accurate strain characterization. This study shows the important associations between cattle and
C. jejuni
clonal complexes ST-61, ST-21, and ST-42, and it suggests that cattle and/or dairy products are likely to be a source of the human
Campylobacter
gastroenteritis caused by such genotypes. The reported findings have significant implications for the design of effective intervention strategies for disease control and prevention.
Effect of Exposure to UV-C Irradiation and Monochloramine on Adenovirus Serotype 2 Early Protein Expression and DNA Replication
Authors: Sirikanchana Kwanrawee , Shisler Joanna L. , Mariñas Benito J.
Published: April 2008

The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20°C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.
Genetic Structure and Biology of
Xylella fastidiosa
Strains Causing Disease in Citrus and Coffee in Brazil
Authors: Almeida Rodrigo P. P. , Nascimento Fernanda E. , Chau John , Prado Simone S. , Tsai Chi-Wei , Lopes Sílvio A. , Lopes Joao R. S.
Published: April 2008

Xylella fastidiosa
is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether
X. fastidiosa
isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing
X. fastidiosa
isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee
X. fastidiosa
isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most
X. fastidiosa
populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of
X. fastidiosa
that have diverged but are genetically homogenized by frequent recombination.
Associations between Antimicrobial Resistance Genes in Fecal Generic
Escherichia coli
Isolates from Cow-Calf Herds in Western Canada
Authors: Gow Sheryl P. , Waldner Cheryl L. , Harel Josee , Boerlin Patrick
Published: April 2008

The objective of this study was to examine associations among the genetic determinants of antimicrobial resistance (AMR) in 207 fecal generic
Escherichia coli
isolates obtained from 77 cow-calf herds in western Canada. Twenty-three resistance genes corresponding to six different antimicrobial families were assessed using DNA hybridization and PCR. The most common resistance genes in the study sample (207 isolates) were
sul2
(48.3%),
tet
(B) (45.4%), and
ant(3
″
)-Ia
(
aadA1
) (19.3%). Several statistically significant associations between the examined resistance genes were detected. The strongest associations observed were those between genes for resistance to chloramphenicol (
catI
) and trimethoprim (
dhfrI
) (odds ratio [OR] = 214;
P
= 0.0001), sulfonamide (
sul1
) and chloramphenicol (
catI
) (OR = 96.9;
P
= 0.0001), streptomycin [
ant(3
″
)-Ia
(
aadA1
)] and trimethoprim (
dhfrI
) (OR = 96.2;
P
= 0.0001), sulfonamide (
sul1
) and streptomycin [
ant(3
″
)-Ia
(
aadA1
)] (OR = 79.3;
P
= 0.0001), and tetracycline [
tet
(B)] and sulfonamides (
sul2
) (OR = 25.7;
P
= 0.0001). At least one of the resistance genes corresponding to each nonaminoglycoside family of antimicrobials examined in this study was associated with the two aminoglycoside resistance genes
ant(3
″
)-Ia
(
aadA1
) and
aph(3
′)-
Ia
. The multiple, strong associations between genes and the diverse nature of the associations described in this study demonstrate the complexity of resistance gene selection in cow-calf herds and should be considered in the planning of AMR control practices for cow-calf operations.
Influence of Environmental Stress on Distributions of Times to First Division in
Escherichia coli
Populations, as Determined by Digital-Image Analysis of Individual Cells
Authors: Niven Gordon W. , Morton Jennifer S. , Fuks Tamara , Mackey Bernard M.
Published: April 2008

The distributions of times to first cell division were determined for populations of
Escherichia coli
stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the “box area ratio.” Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37°C and inoculated on agar and incubated at 20°C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50°C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The “relative division time,” which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the “work to be done” to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.
Anaerobic and Aerobic Degradation of Cyanophycin by the Denitrifying Bacterium
Pseudomonas alcaligenes
Strain DIP1 and Role of Three Other Coisolates in a Mixed Bacterial Consortium
Authors: Sallam Ahmed , Steinbüchel Alexander
Published: April 2008

Four bacterial strains were isolated from a cyanophycin granule polypeptide (CGP)-degrading anaerobic consortium, identified by 16S rRNA gene sequencing, and assigned to species of the genera
Pseudomonas
,
Enterococcus
,
Clostridium
, and
Paenibacillus
. The consortium member responsible for CGP degradation was assigned as
Pseudomonas alcaligenes
strain DIP1. The growth of and CGP degradation by strain DIP1 under anaerobic conditions were enhanced but not dependent on the presence of nitrate as an electron acceptor. CGP was hydrolyzed to its constituting β-Asp-Arg dipeptides, which were then completely utilized within 25 and 4 days under anaerobic and aerobic conditions, respectively. The end products of CGP degradation by strain DIP1 were alanine, succinate, and ornithine as determined by high-performance liquid chromatography analysis. The facultative anaerobic
Enterococcus casseliflavus
strain ELS3 and the strictly anaerobic
Clostridium sulfidogenes
strain SGB2 were coisolates and utilized the β-linked isodipeptides from the common pool available to the mixed consortium, while the fourth isolate,
Paenibacillus odorifer
strain PNF4, did not play a direct role in the biodegradation of CGP. Several syntrophic interactions affecting CGP degradation, such as substrate utilization, the reduction of electron acceptors, and aeration, were elucidated. This study demonstrates the first investigation of CGP degradation under both anaerobic and aerobic conditions by one bacterial strain, with regard to the physiological role of other bacteria in a mixed consortium.
Chitin Hydrolysis by
Listeria
spp., Including
L. monocytogenes
Authors: Leisner J. J. , Larsen M. H. , Jørgensen R. L. , Brøndsted L. , Thomsen L. E. , Ingmer H.
Published: April 2008

Listeria
spp., including the food-borne pathogen
Listeria monocytogenes
, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear β-1,4-linked polymer of
N
-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that
L. monocytogenes
and other
Listeria
spp. are able to hydrolyze α-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30°C than at 37°C. In
L. monocytogenes
EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (
chiB
) and lmo1883 (
chiA
), but not on a gene encoding a putative chitin binding protein (lmo2467). The
chiB
and
chiA
genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of
Listeria
are discussed.
Minimal
Escherichia coli
Cell for the Most Efficient Production of Ethanol from Hexoses and Pentoses
Authors: Trinh Cong T. , Unrean Pornkamol , Srienc Friedrich
Published: April 2008

To obtain an efficient ethanologenic
Escherichia coli
strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Elimination of three additional genes resulted in a strain that selectively grows only on pentoses, even in the presence of glucose, with a high ethanol yield. We showed that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions. Remarkably, catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production.
The Cyclic AMP-Dependent Catabolite Repression System of
Serratia marcescens
Mediates Biofilm Formation through Regulation of Type 1 Fimbriae
Authors: Kalivoda Eric J. , Stella Nicholas A. , O'Dee Dawn M. , Nau Gerard J. , Shanks Robert M. Q.
Published: April 2008

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in
Serratia marcescens
biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of
S. marcescens
in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in
Escherichia coli
and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (
cyaA
) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (
crr
and
cyaA
) or the ability to respond to cAMP (
crp
) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (
crp
) mutant require the
fimABCD
operon, which is responsible for type 1 fimbria production. Consistently,
fimA
transcription and fimbria production were determined to be upregulated in a
cyaA
mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development.
Development and Experimental Validation of a Predictive Threshold Cycle Equation for Quantification of Virulence and Marker Genes by High-Throughput Nanoliter-Volume PCR on the OpenArray Platform
Authors: Stedtfeld Robert D. , Baushke Samuel W. , Tourlousse Dieter M. , Miller Sarah M. , Stedtfeld Tiffany M. , Gulari Erdogan , Tiedje James M. , Hashsham Syed A.
Published: April 2008

Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (

C
T

) equation was developed to calculate the number of starting copies based on an experimental

C
T

. The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (
R
2
= 0.816) was observed between the predicted and experimental

C
T

s based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3′ end. The performance of the predictive

C
T

equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive

C
T

equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.
Competing Isogenic
Campylobacter
Strains Exhibit Variable Population Structures In Vivo
Authors: Coward Chris , van Diemen Pauline M. , Conlan Andrew J. K. , Gog Julia R. , Stevens Mark P. , Jones Michael A. , Maskell Duncan J.
Published: April 2008

Consumption of poultry contaminated with
Campylobacter jejuni
is a risk factor for human gastrointestinal disease. The rational development of control strategies for
Campylobacter
within chickens requires an understanding of the colonization process at the molecular and population levels, both within and between hosts. Experiments employing competing strains of
Campylobacter
have been used to investigate colonization. Implicit in these studies is the assumption that the behavior of competing strains is reproducible between experiments. Variability in the recovery of mutants from the chicken gastrointestinal tract during signature-tagged mutagenesis studies demonstrated that this is not always the case. To further investigate this phenomenon in the absence of confounding factors due to phenotypic differences between mutants, we constructed individually identifiable
w
ild-type
i
sogenic
t
agged
s
trains (WITS) that have indistinguishable phenotypes in pure culture. By using mixtures of WITS, it is possible to monitor the relative amounts of subpopulations of essentially wild-type bacteria. Using a 2-week-old chicken model of colonization, we observed unpredictable variations in population structure both within and between experiments, even in the simplest case of two competing strains. This variation occurred both when birds were simultaneously infected with two WITS and when birds inoculated with different WITS were cohoused. We present evidence for founder effects during initial colonization with subsequent bird-to-bird transmission. We suggest that these and phenotypic variation contribute to the observed variability. These factors render simple models of colonization which do not take them into account inappropriate for
Campylobacter
and impact the planning and interpretation of competition experiments using this organism.
Rapid Screening of Quorum-Sensing Signal
N
-Acyl Homoserine Lactones by an In Vitro Cell-Free Assay
Authors: Kawaguchi Tomohiro , Chen Yung Pin , Norman R. Sean , Decho Alan W.
Published: April 2008

A simple, sensitive, and rapid cell-free assay system was developed for detection of
N
-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium
Agrobacterium tumefaciens
NTL4(pCF218)(pCF372), allowing the expression of β-galactosidase upon addition of exogenous AHLs. We have shown that β-galactosidase expression is possible in cell-free solution [lysate from
Agrobacterium tumefaciens
NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (−80°C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.
Construction of a Reporter Vector System for In Vivo Analysis of Promoter Activity in
Propionibacterium freudenreichii
Authors: Faye Therese , Åsebø Anita , Salehian Zhian , Langsrud Thor , Nes Ingolf F. , Brede Dag Anders
Published: April 2008

A β-galactosidase reporter system for the analysis of promoter elements in
Propionibacterium freudenreichii
was designed. The pTD210 in vivo reporter vector was constructed using a promoterless
lacZ
gene from
Bifidobacterium longum
cloned into the pAMT1 plasmid. The utility of the pTD210 reporter vector was demonstrated by an investigation of six predicted promoters in
P. freudenreichii
. The system produced accurate and reproducible measurements that facilitated both promoter identification and the quantification of promoter activities.
Characterization of a Bacterial Community in an Abandoned Semiarid Lead-Zinc Mine Tailing Site
Authors: Mendez Monica O. , Neilson Julia W. , Maier Raina M.
Published: April 2008

Bacterial diversity in mine tailing microbial communities has not been thoroughly investigated despite the correlations that have been observed between the relative microbial diversity and the success of revegetation efforts at tailing sites. This study employed phylogenetic analyses of 16S rRNA genes to compare the bacterial communities present in highly disturbed, extremely (pH 2.7) and moderately (pH 5.7) acidic lead-zinc mine tailing samples from a semiarid environment with those from a vegetated off-site (OS) control sample (pH 8). Phylotype richness in these communities decreased from 42 in the OS control to 24 in the moderately acidic samples and 8 in the extremely acidic tailing samples. The clones in the extremely acidic tailing sample were most closely related to acidophiles, none of which were detected in the OS control sample. The comparison generated by this study between the bacteria present in extremely acidic tailing and that in moderately acidic tailing communities with those in an OS control soil provides a reference point from which to evaluate the successful restoration of mine tailing disposal sites by phytostabilization.
Biphenyl Dioxygenase from an Arctic Isolate Is Not Cold Adapted
Authors: Master Emma R. , Agar Nathalie Y. R. , Gómez-Gil Leticia , Powlowski Justin B. , Mohn William W. , Eltis Lindsay D.
Published: April 2008

Biphenyl dioxygenase from the psychrotolerant bacterium
Pseudomonas
sp. strain Cam-1 (BPDO
Cam-1
) was purified and found to have an apparent

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for biphenyl of 1.1 ± 0.1 s
−1
(mean ± standard deviation) at 4°C. In contrast, BPDO
LB400
from the mesophile
Burkholderia xenovorans
LB400 had no detectable activity at this temperature. At 57°C, the half-life of the BPDO
Cam-1
oxygenase was less than half that of the BPDO
LB400
oxygenase. Nevertheless, BPDO
Cam-1
appears to be a typical
Pseudomonas pseudoalcaligenes
KF707-type dioxygenase.
Candida albicans
Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction
Authors: Kebaara Bessie W. , Langford Melanie L. , Navarathna Dhammika H. M. L. P. , Dumitru Raluca , Nickerson Kenneth W. , Atkin Audrey L.
Published: April 2008

Candida albicans
is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The
tup1/tup1
and
nrg1/nrg1
mutants, but not the
rfg1/rfg1
mutant, failed to respond to farnesol. Treatment of
C. albicans
cells with farnesol caused a small but consistent increase in both
TUP1
mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes,
HWP1
and
RBT1
. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a
TUP1/tup1
heterozygote. Farnesol did not affect
EFG1
(a transcription regulator of filament development),
NRG1
, or
RFG1
mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the
tup1/tup1
and
nrg1/nrg1
mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in
C. albicans
.
Transcriptional Interference and Repression Modulate the Conjugative Ability of the Symbiotic Plasmid of
Rhizobium etli
Authors: Sepúlveda Edgardo , Pérez-Mendoza Daniel , Ramírez-Romero Miguel A. , Soto María J. , López-Lara Isabel M. , Geiger Otto , Sanjuán Juan , Brom Susana , Romero David
Published: April 2008

Bacteria of the order
Rhizobiales
are able to establish nitrogen-fixing symbioses with legumes. Commonly, genes for symbiosis are harbored on large symbiotic plasmids. Although the transfer of symbiotic plasmids is commonly detected in nature, there are few experimentally characterized examples. In
Rhizobium etli
, the product of
rctA
inhibits the conjugation of the symbiotic plasmid by reducing the transcription of the
virB
operon.
rctA
is transcribed divergently from this operon, and its product is predicted to have a DNA binding domain. In the present study, using DNase I footprinting and binding assays, we demonstrated the specific binding of RctA to the
virB
operon promoter. A 9-bp motif in the spacer region of this promoter (the
rctA
binding motif box) and the presence of a functional −10 region were critical elements for RctA binding. Transcriptional fusion analyses revealed that the elimination of either element provoked a relief of RctA-mediated repression. These data support a model in which RctA inhibits the access of the RNA polymerase to the
virB
promoter. Interestingly,
rctA
expression levels were modulated by transcriptional interference from transcripts emanating from the
virB
promoter. This phenomenon adds another level of regulation for this system, thus revealing a novel mechanism of plasmid transfer regulation in the
Rhizobiales
.
Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ
Authors: Cocozaki Alexis I. , Ghattas Ingrid R. , Smith Colin A.
Published: April 2008

Transcription antitermination in phages λ and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the λ N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB
left
complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and λ N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by λ N. Point mutations can dramatically alter boxB specificity between P22 and λ N. A boxB specific for P22 N can be mutated to λ N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.
UpaG, a New Member of the Trimeric Autotransporter Family of Adhesins in UropathogenicEscherichia coli
Authors: Valle Jaione , Mabbett Amanda N. , Ulett Glen C. , Toledo-Arana Alejandro , Wecker Karine , Totsika Makrina , Schembri Mark A. , Ghigo Jean-Marc , Beloin Christophe
Published: April 2008

The ability ofEscherichia colito colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenicE. coli(UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed thatupaGis strongly associated withE. colistrains from the B2 and D phylogenetic groups, while deletion ofupaGhad no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin fromE. colithat mediates aggregation, biofilm formation, and adhesion to various ECM proteins.
Formate Acts as a Diffusible Signal To InduceSalmonellaInvasion
Authors: Huang Yanyan , Suyemoto Mitsu , Garner Cherilyn D. , Cicconi Kellie M. , Altier Craig
Published: April 2008

To infect an animal host,Salmonella entericaserovar Typhimurium must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded withinSalmonellapathogenicity island I (SPI1). We found that a mutant with deletions of the acetate kinase and phosphotransacetylase genes (ackA-pta) was deficient in invasion and SPI1 expression but that invasion gene expression was completely restored by supplying medium conditioned by growth of the wild-type strain, suggesting that a signal produced by the wild type, but not by theackA-ptamutant, was required for invasion. This mutant also excreted 68-fold-less formate into the culture medium, and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH dependent, requiring a pH below neutrality, and studies in mice showed that the distal ileum, the preferred site ofSalmonellainvasion in this species, had the appropriate formate concentration and pH to elicit invasion, while the cecum contained no detectable formate. Furthermore, we found that formate affected the major regulators of SPI1,hilAandhilD, but that the primary routes of formate metabolism played no role in its activity as a signal.
Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of
Escherichia coli
Authors: Sperandeo Paola , Lau Fion K. , Carpentieri Andrea , De Castro Cristina , Molinaro Antonio , Dehò Gianni , Silhavy Thomas J. , Polissi Alessandra
Published: April 2008

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.
Conserved Residues Asp16 and Pro24 of TnaC-tRNA
Pro
Participate in Tryptophan Induction of
tna
Operon Expression
Authors: Cruz-Vera Luis R. , Yanofsky Charles
Published: April 2008

In
Escherichia coli
, interactions between the nascent TnaC-tRNA
Pro
peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA
Pro
cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased
tna
operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of
tna
operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of
tna
operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in
tna
operon induction. Replacing Asp16 or Pro24 of TnaC of
E. coli
with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA
Pro
cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA
Pro
peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.
The ArgP Protein Stimulates the
Klebsiella pneumoniae gdhA
Promoter in a Lysine-Sensitive Manner
Authors: Goss Thomas J.
Published: April 2008

The lysine-sensitive factor that binds to the upstream region of the
Klebsiella pneumoniae gdhA
promoter and stimulates
gdhA
transcription during growth in minimal medium has been proposed to be the
K. pneumoniae
ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the
K. pneumoniae argP
gene was generated and used to assess the roles of exogenous lysine and
argP
in the regulation of the
gdhA
promoter. Disruption of
argP
reduced the strength and the lysine-dependent regulation of the
gdhA
promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and
argP
-defective strains indicted the presence of an
argP
-dependent factor whose ability to bind the
gdhA
promoter was lysine sensitive. DNase I footprinting studies using purified
K. pneumoniae
ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the
gdhA
transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the
gdhA
promoter region in vitro and the
argP
-dependent stimulation of the
gdhA
promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the
gdhA
promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.
YscP and YscU Switch the Substrate Specificity of the
Yersinia
Type III Secretion System by Regulating Export of the Inner Rod Protein YscI
Authors: Wood Sarah E. , Jin Jin , Lloyd Scott A.
Published: April 2008

Pathogenic yersiniae utilize a type III secretion system to inject antihost factors, called Yops, directly into the cytosol of eukaryotic cells. The Yops are injected via a needle-like structure, comprising the YscF protein, on the bacterial surface. While the needle is being assembled, Yops cannot be secreted. YscP and YscU switch the substrate specificity of the secretion system to enable Yop export once the needle attains its proper length. Here, we demonstrate that the inner rod protein YscI plays a critical role in substrate specificity switching. We show that YscI is secreted by the type III secretion system and that YscI secretion by a
yscP
mutant is abnormally elevated. Furthermore, we show that mutations in the cytoplasmic domain of YscU reduce YscI secretion by the
yscP
null strain. We also demonstrate that mutants expressing one of three forms of YscI (those with mutations Q84A, L87A, and L96A) secrete substantial amounts of Yops yet exhibit severe defects in needle formation. In the absence of YscP, mutants with the same changes in YscI assemble needles but are unable to secrete Yops. Together, these results suggest that the formation of the inner rod, not the needle, is critical for substrate specificity switching and that YscP and YscU exert their effects on substrate export by controlling the secretion of YscI.
ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of
Bacillus cereus
Authors: Esbelin Julia , Jouanneau Yves , Armengaud Jean , Duport Catherine
Published: April 2008

Bacillus cereus
Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of
hbl
and
nhe
enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes.
B. cereus
Fnr was overexpressed and purified as either a C-terminal His-tagged (Fnr
His
) fusion protein or an N-terminal fusion protein tagged with the
Strep
-tag (IBA BioTAGnology) (
Strep
Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr
His
was mainly monomeric, while apo
Strep
Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo
Strep
Fnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators
fnr
,
resDE
, and
plcR
and the structural genes
hbl
and
nhe
. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.
The Critical Role ofembCinMycobacterium tuberculosis
Authors: Goude Renan , Amin Anita G. , Chatterjee Delphi , Parish Tanya
Published: April 2008

Arabinan polymers are major components of the cell wall inMycobacterium tuberculosisand are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic speciesMycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and anembCmutant is viable but lacks LAM. In contrast, we demonstrate here that inM. tuberculosis,embCis an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria.M. tuberculosisEmbC has an activity similar to that ofM. smegmatisEmbC, since we were able to complement anembCmutant ofM. smegmatiswithembCMtb, confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced inM. smegmatiswas dependent on the level of expression ofembCMtb. Northern analysis revealed thatembCis expressed as part of a polycistronic message encompassingembCand three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis inM. tuberculosisand confirmed its essential role in this species.
Identification of a Novel Trimeric Autotransporter Adhesin in the Cryptic Genospecies of
Haemophilus
Authors: Sheets Amanda J. , Grass Susan A. , Miller Sara E. , St. Geme Joseph W.
Published: April 2008

Haemophilus
biotype IV strains belonging to the recently recognized
Haemophilus
cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of
Haemophilus
cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for
c
ryptic
h
aemophilus
a
dhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the
Haemophilus
cryptic genospecies and is absent from the closely related species
Haemophilus influenzae
. We speculate that Cha is important in the pathogenesis of disease due to the
Haemophilus
cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.
Mutations in thetacFGene of Clinical Strains and Laboratory Transformants ofStreptococcus pneumoniae:Impact on Choline Auxotrophy and Growth Rate
Authors: González Ana , Llull Daniel , Morales María , García Pedro , García Ernesto
Published: April 2008

The nutritional requirement thatStreptococcus pneumoniaehas for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in thetacFgene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype ofS. pneumoniae(M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report thatStreptococcus mitiscan grow in choline-free medium, as previously illustrated forStreptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation ofS. pneumoniaeR6 by usingS. mitisSK598 DNA and several PCR-amplifiedtacFfragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independentS. pneumoniaemutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant ofS. pneumoniae(strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for thetacFgene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.