bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and Replication of the Pathogenicity Plasmid pPATH in Diverse Populations of the Gall-Forming Bacterium
Pantoea agglomerans
Authors: Weinthal Dan M. , Barash Isaac , Panijel Mary , Valinsky Lea , Gaba Victor , Manulis-Sasson Shulamit
Published: October 2007

Pantoea agglomerans
has been transformed from a commensal bacterium into two related gall-forming pathovars by acquisition of pPATH plasmids containing a pathogenicity island (PAI). This PAI harbors an
hrp/hrc
gene cluster, type III effectors, and phytohormone biosynthetic genes. DNA typing by pulsed-field gel electrophoresis revealed two major groups of
P. agglomerans
pv. gypsophilae and one group of
P. agglomerans
pv. betae. The pPATH plasmids of the different groups had nearly identical replicons (98% identity), and the RepA protein showed the highest level of similarity with IncN plasmid proteins. A series of plasmids, designated pRAs, in which the whole replicon region (2,170 bp) or deleted derivatives of it were ligated with
nptI
were generated for replicon analysis. A basic 929-bp replicon (pRA6) was sufficient for replication in
Escherichia coli
and in nonpathogenic
P. agglomerans
. However, the whole replicon region (pRA1) was necessary for expulsion of the pPATH plasmid, which resulted in the loss of pathogenicity. The presence of direct repeats in the replicon region suggests that the pPATH plasmid is an iteron plasmid and that the repeats may regulate its replication. The pPATH plasmids are nonconjugative but exhibit a broad host range, as shown by replication of pRA1 in
Erwinia, Pseudomonas
, and
Xanthomonas
. Restriction fragment length polymorphism analyses indicated that the PAIs in the two groups of
P. agglomerans
pv. gypsophilae are similar but different from those in
P. agglomerans
pv. betae. The results could indicate that the pPATH plasmids evolved from a common ancestral mobilizable plasmid that was transferred into different strains of
P. agglomerans
.
The Cyclic AMP Receptor Protein Modulates Colonial Morphology in
Vibrio cholerae
Authors: Liang Weili , Silva Anisia J. , Benitez Jorge A.
Published: October 2007

Inactivation of the quorum-sensing regulator HapR causes
Vibrio cholerae
El Tor biotype strain C7258 to adopt a rugose colonial morphology that correlates with enhanced biofilm formation.
V. cholerae
mutants lacking the cyclic AMP (cAMP) receptor protein (CRP) produce very little HapR, which results in elevated expression of
Vibrio
exopolysaccharide (
vps
) genes and biofilm compared to the wild type. However, Δ
crp
mutants still exhibited smooth colonial morphology and expressed reduced levels of
vps
genes compared to isogenic
hapR
mutants. In this study we demonstrate that deletion of
crp
and
cya
(adenylate cyclase) converts a rugose Δ
hapR
mutant to a smooth one. The smooth Δ
hapR
Δ
crp
and Δ
hapR
Δ
cya
double mutants could be converted back to rugose by complementation with
crp
and
cya
, respectively. CRP was found to enhance the expression of VpsR, a strong activator of
vps
expression, but to diminish transcription of VpsT. Ectopic expression of VpsR in smooth Δ
hapR
Δ
crp
and Δ
hapR
Δ
cya
double mutants restored rugose colonial morphology. Lowering intracellular cAMP levels in a Δ
hapR
mutant by the addition of glucose diminished VpsR expression and colonial rugosity. On the basis of our results, we propose a model for the regulatory input of CRP on exopolysaccharide biosynthesis.
Epidemiology, Relative Invasive Ability, Molecular Characterization, and Competitive Performance of
Campylobacter jejuni
Strains in the Chicken Gut
Authors: Pope Christopher , Wilson Janet , Taboada Eduardo N. , MacKinnon Joanne , Felipe Alves Cristiano A. , Nash John H. E. , Rahn Kris , Tannock Gerald W.
Published: October 2007

One hundred forty-one
Campylobacter jejuni
isolates from humans with diarrhea and 100 isolates from retailed poultry meat were differentiated by
flaA
typing. The bacteria were isolated in a specific geographical area (Dunedin) in New Zealand over a common time period. Twenty nine
flaA
types were detected, one of which (
flaA
restriction fragment length polymorphism type 15 [
flaA
-15]) predominated among isolates from humans (∼30% of isolates). This strain was of low prevalence (5% of isolates) among poultry isolates.
flaA
-15 strains were five to six times more invasive of HEp2 cells in an in vitro assay than a
flaA
type (
flaA
-3) that was commonly encountered on poultry meat (23% of isolates) but was seldom associated with human illness (5%). Competitive-exclusion experiments with chickens, utilizing real-time quantitative PCR to measure the population sizes of specific strains representing
flaA
-15 (T1016) and
flaA
-3 (Pstau) in digesta, were carried out. These experiments showed that T1016 always outcompeted Pstau in the chicken intestine. Genomic comparisons of T1016 and Pstau were made using DNA microarrays representing the genome of
C. jejuni
NCTC 11168. These comparisons revealed differences between the strains in the gene content of the Cj1417c-to-Cj1442c region of the genome, which is associated with the formation of capsular polysaccharide. The strains differed in Penner type (T1016, O42; Pstau, O53). It was concluded that poultry meat was at least one source of human infection with
C. jejuni
, that some
Campylobacter
strains detected in poultry meat are of higher virulence for humans than others, and that bacterial attributes affecting strain virulence and commensal colonization ability may be linked.
Variability of Total and PathogenicVibrio parahaemolyticusDensities in Northern Gulf of Mexico Water and Oysters
Authors: Zimmerman A. M. , DePaola A. , Bowers J. C. , Krantz J. A. , Nordstrom J. L. , Johnson C. N. , Grimes D. J.
Published: October 2007

Vibrio parahaemolyticusis indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies ofV. parahaemolyticushave identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenicV. parahaemolyticusorganisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P< 0.001) between totalV. parahaemolyticusdensities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. PathogenicV. parahaemolyticusorganisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenicV. parahaemolyticusdensities than in totalV. parahaemolyticusdensities. These data suggest that, although totalV. parahaemolyticusdensities may be very informative, there is greater uncertainty when totalV. parahaemolyticusdensities are used to predict the risk of infection by pathogenicV. parahaemolyticusthan previously recognized.
Improvement of NADPH-Dependent Bioconversion by Transcriptome-Based Molecular Breeding
Authors: Hibi Makoto , Yukitomo Hiromi , Ito Mikito , Mori Hideo
Published: October 2007

Transcriptome data for a xylitol-producing recombinant
Escherichia coli
were obtained and used to tune up its productivity. Structural genes of NADPH-dependent
d
-xylose reductase and
d
-xylose permease were inserted into an
Escherichia coli
chromosome to construct a recombinant strain producing xylitol from
d
-xylose for use as a model system for NADPH-dependent bioconversion. Transcriptome analysis of xylitol-producing and nonproducing conditions for the recombinant revealed that xylitol production down-regulated 56 genes. These genes were then selected as candidate factors for suppression of the NADPH supply and were disrupted to validate their functions. Of the gene disruptants, that resulting from the deletion of
yhbC
showed the best bioconversion rate. Also, the deletion accelerated cell growth during log phase. The features of the mutant could be maintained in jar fermenter-scale production of xylitol. Thus, our novel molecular host strain breeding method using transcriptome analysis was fully effective and could be applied to improving various industrial strains.
Genomic and Biochemical Studies Demonstrating the Absence of an Alkane-Producing Phenotype in
Vibrio furnissii
M1
Authors: Wackett Lawrence P. , Frias Janice A. , Seffernick Jennifer L. , Sukovich David J. , Cameron Stephan M.
Published: October 2007

Vibrio furnissii
M1 was recently reported to biosynthesize
n
-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol.
187:
1426-1429, 2005). In the present study,
V. furnissii
M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that
V. furnissii
M1 was not identical to other
V. furnissii
strains tested, but the level of relatedness was consistent with its assignment as a
V. furnissii
strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other
Vibrio
strains. Complete genomic DNA from
V. furnissii
M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover,
V. furnissii
M1 did not produce demonstrable levels of
n
-alkanes in vivo or in vitro. In vivo experiments were conducted by growing
V. furnissii
M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [
14
C]hexadecanol. The data are consistent with the present strain being a
V. furnissii
with properties similar to those previously described but lacking the alkane-producing phenotype.
V. furnissii
ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.
Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus
Authors: Nielsen Michael L. , de Jongh Willem A. , Meijer Susan L. , Nielsen Jens , Mortensen Uffe H.
Published: October 2007

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of
pyrG
. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed,
pyrG
function can be rapidly returned to wild type by a simple selection scheme.
Trypanosoma brucei
RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway
Authors: Hellman Kristina , Prohaska Kimberly , Williams Noreen
Published: October 2007

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in
Trypanosoma brucei
. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.
Endocytosis in the Shiitake Mushroom
Lentinula edodes
and Involvement of GTPase LeRAB7
Authors: Lee Ming Tsung , Szeto Carol Ying Ying , Ng Tak Pan , Kwan Hoi Shan
Published: October 2007

Endocytosis is the process by which substrates enter a cell without passing through the plasma membrane but rather invaginate the cell membrane and form intracellular vesicles.
Rab7
regulates endocytic trafficking between early and late endosomes and between late endosomes and lysosomes. Le
Rab7
in
Lentinula edodes
is strongly homologous to
Rab7
in
Homo sapiens
. Receptors for activated C kinase-1 (LeRACK1) and Rab5 GTPase (LeRAB5) were isolated as interacting partners of LeRab7, and the interactions were confirmed by in vivo and in vitro protein interaction assays. The three genes showed differential expression in the various developmental stages of the mushroom. In situ hybridization showed that the three transcripts were localized in regions of active growth, such as the outer region of trama cells, and the subhymenium of the hymenophore of mature fruiting bodies and the prehymenophore of young fruiting bodies. The existence of endocytosis in the mycelium and hymenophores was confirmed by the internalization of FM4-64. LeRAB7 was partially colocalized with the AM4-64 and was located in the late endocytic pathway. This is the first report of the presence of endocytosis in homobasidiomycetes. LeRAB7, LeRAB5, and LeRACK1 may contribute to the growth of
L. edodes
and cell differentiation in hymenophores.
Unexpected Link between Metal Ion Deficiency and Autophagy in
Aspergillus fumigatus
Authors: Richie Daryl L. , Fuller Kevin K. , Fortwendel Jarrod , Miley Michael D. , McCarthy Jason W. , Feldmesser Marta , Rhodes Judith C. , Askew David S.
Published: October 2007

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus
Aspergillus fumigatus
, we disrupted the
A. fumigatus atg1
gene, encoding a serine/threonine kinase required for autophagy. The ΔAf
atg1
mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAf
atg1
mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAf
atg1
mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type
A. fumigatus
, even in the presence of abundant carbon and nitrogen, and the ΔAf
atg1
mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.
The
Aspergillus fumigatus
Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model
Authors: Lessing Franziska , Kniemeyer Olaf , Wozniok Iwona , Loeffler Juergen , Kurzai Oliver , Haertl Albert , Brakhage Axel A.
Published: October 2007

Macrophages and neutrophils kill the airborne fungal pathogen
Aspergillus fumigatus
. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of
A. fumigatus
challenged by H
2
O
2
. Since many of the identified proteins and genes are apparently regulated by a putative
Saccharomyces cerevisiae
Yap1 homolog, the corresponding gene of
A. fumigatus
was identified and designated Af
yap1
. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Af
yap1
gene led to drastically increased sensitivity of the deletion mutant against H
2
O
2
and menadione, but not against diamide and NO radicals. Proteome analysis of the ΔAf
yap1
mutant strain challenged with 2 mM H
2
O
2
indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Af
yap1
deletion mutant did not show attenuated virulence in a murine model of
Aspergillus
infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of
A. fumigatus
in an immunocompromised host. This conclusion was further supported by the finding that killing of
A. fumigatus
wild-type and ΔAf
yap1
mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.
Expression and Characterization of the Flocculin Flo11/Muc1, a
Saccharomyces cerevisiae
Mannoprotein with Homotypic Properties of Adhesion
Authors: Douglas Lois M. , Li Li , Yang Yang , Dranginis A. M.
Published: October 2007

The Flo11/Muc1 flocculin has diverse phenotypic effects.
Saccharomyces cerevisiae
cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that
S. cerevisiae
var.
diastaticus
strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to
S. cerevisiae
var.
diastaticus
cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to
S. cerevisiae
var.
diastaticus
cells expressing
FLO11
and failed to bind to cells with a deletion of
FLO11
, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.
Functional Analysis of the M.HpyAIV DNA Methyltransferase of
Helicobacter pylori
Authors: Skoglund Anna , Björkholm Britta , Nilsson Christina , Andersson Anders F. , Jernberg Cecilia , Schirwitz Katja , Enroth Cristofer , Krabbe Margareta , Engstrand Lars
Published: October 2007

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen
Helicobacter pylori
. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the
H. pylori
strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase.
H. pylori
strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from
H. pylori
strain 26695 was overexpressed in
Escherichia coli
, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of
H. pylori
strains 26695 and J99 resulted in a number of candidate genes.
katA
, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.
Response of
Desulfovibrio vulgaris
to Alkaline Stress
Authors: Stolyar Sergey , He Qiang , Joachimiak Marcin P. , He Zhili , Yang Zamin Koo , Borglin Sharon E. , Joyner Dominique C. , Huang Katherine , Alm Eric , Hazen Terry C. , Zhou Jizhong , Wall Judy D. , Arkin Adam P. , Stahl David A.
Published: October 2007

The response of exponentially growing
Desulfovibrio vulgaris
Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of
D. vulgaris
to increased pH is generally similar to that of
Escherichia coli
but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in
E. coli
appear to be absent in
D. vulgaris
. Genes previously reported to be up-regulated in
E. coli
were up-regulated in
D. vulgaris
; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in
D. vulgaris
. As in
E. coli
, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a
D. vulgaris
Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).
Characterization of the
Mycobacterium tuberculosis
4-Diphosphocytidyl-2-
C
-Methyl-
d
-Erythritol Synthase: Potential for Drug Development
Authors: Eoh Hyungjin , Brown Amanda C. , Buetow Lori , Hunter William N. , Parish Tanya , Kaur Devinder , Brennan Patrick J. , Crick Dean C.
Published: October 2007

Mycobacterium tuberculosis
utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for
Escherichia coli
. In the MEP pathway, 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol is formed from 2-
C
-methyl-
d
-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for
M. tuberculosis
: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified
M. tuberculosis
IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-
C
-methyl-
d
-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg
2+
being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested,
M. tuberculosis
IspD had

K
m

values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated
k
cat
and
k
cat
/

K
m

values were 0.72 min
−1
and 12.3 mM
−1
min
−1
for MEP and 1.0 min
−1
and 18.8 mM
−1
min
−1
for CTP, respectively.
Multiple Superoxide Dismutases in
Agrobacterium tumefaciens
: Functional Analysis, Gene Regulation, and Influence on Tumorigenesis
Authors: Saenkham Panatda , Eiamphungporn Warawan , Farrand Stephen K. , Vattanaviboon Paiboon , Mongkolsuk Skorn
Published: October 2007

Agrobacterium tumefaciens
possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme.
sodBI
is expressed at a high levels throughout all growth phases.
sodBII
expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner.
sodBIII
is expressed only during stationary phase. Analysis of the physiological function of
sod
s reveals that the inactivation of
sodBI
markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all
sod
s contribute at various levels towards the overall menadione resistance level. Sods also have important roles in
A. tumefaciens
virulence toward a host plant. A
sodBI
but not a
sodBII
or
sodBIII
mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple
sod
null mutant is avirulent.
Comparative Genomic Hybridization Detects Secondary Chromosomal Deletions in
Escherichia coli
K-12 MG1655 Mutants and Highlights Instability in the
flhDC
Region
Authors: Hobman Jon L. , Patel Mala D. , Hidalgo-Arroyo G. Aida , Cariss S. James L. , Avison Matthew B. , Penn Charles W. , Constantinidou Chrystala
Published: October 2007

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in
Escherichia coli
K-12 MG1655, we identified unintended secondary genomic deletions in the
flhDC
region in Δ
fnr
, Δ
crp
, and Δ
creB
mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.
A Conserved Glycine Residue of Trimeric Autotransporter Domains Plays a Key Role inYersiniaAdhesin A Autotransport
Authors: Grosskinsky Ulrike , Schütz Monika , Fritz Michaela , Schmid Yvonne , Lamparter Marina C. , Szczesny Pawel , Lupas Andrei N. , Autenrieth Ingo B. , Linke Dirk
Published: October 2007

TheYersiniaadhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA inEscherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in anE. coli degPdeletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.
Spatial Organization ofMyxococcus xanthusduring Fruiting Body Formation
Authors: Curtis Patrick D. , Taylor Rion G. , Welch Roy D. , Shimkets Lawrence J.
Published: October 2007

Microcinematography was used to examine fruiting body development ofMyxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. ApilA(MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. AcsgA(MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.
Identification and Characterization of the Flavin:NADH Reductase (PrnF) Involved in a Novel Two-Component Arylamine Oxygenase
Authors: Lee Jung-Kul , Zhao Huimin
Published: October 2007

Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family (J. Lee et al., J. Biol. Chem.
280
:36719-36728, 2005). Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin
N
-oxygenase (PrnD) represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date. Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (PrnF) involved in the PrnD-catalyzed unusual arylamine oxidation. The
prnF
gene was identified via sequence analysis of the whole genome of
Pseudomonas fluorescens
Pf-5 and subsequently cloned and overexpressed in
Escherichia coli
. The purified PrnF protein catalyzes reduction of flavin adenine dinucleotide (FAD) by NADH with a
k
cat
of 65 s
−1
(
K

m

= 3.2 μM for FAD and 43.1 μM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.
Control of the Transcription of a Short Gene Encoding a Cyclic Peptide in
Streptococcus thermophilus
: a New Quorum-Sensing System?
Authors: Ibrahim Mariam , Guillot Alain , Wessner Francoise , Algaron Florence , Besset Colette , Courtin Pascal , Gardan Rozenn , Monnet Véronique
Published: October 2007

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by
Streptococcus thermophilus
, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three
S. thermophilus
strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by
S. thermophilus
LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.
Evolution and Functional Characterization of the
RH50
Gene from the Ammonia-Oxidizing Bacterium
Nitrosomonas europaea
Authors: Cherif-Zahar Baya , Durand Anne , Schmidt Ingo , Hamdaoui Nabila , Matic Ivan , Merrick Mike , Matassi Giorgio
Published: October 2007

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an
RH50
gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium
Nitrosomonas europaea
. Here we have used a phylogenetic approach to study the evolution of the
N. europaea RH50
gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the
N. europaea
genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50

Ne

protein and the corresponding knockout mutant, we determined that
Ne
Rh50 can mediate ammonium uptake. The

RH50
Ne

gene may thus have replaced functionally the
AMT
gene, which is missing in the genome of
N. europaea
and may be regarded as a case of nonorthologous gene displacement.
Rhodobacter capsulatus
OlsA Is a Bifunctional Enyzme Active in both Ornithine Lipid and Phosphatidic Acid Biosynthesis
Authors: Aygun-Sunar Semra , Bilaloglu Rahmi , Goldfine Howard , Daldal Fevzi
Published: October 2007

The
Rhodobacter capsulatus
genome contains three genes (
olsA
[
plsC138
],
plsC316
, and
plsC3498
) that are annotated as lysophosphatidic acid (1-acyl-
sn
-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes,
olsA
was previously shown to be an
O
-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of
c
-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining
plsC316
and
plsC3498
genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the
Escherichia coli plsC
, neither
plsC316
nor
plsC3498
was essential in
R. capsulatus
. In contrast, no
plsC316 olsA
double mutant could be isolated, indicating that an intact copy of either
olsA
or
plsC316
was required for
R. capsulatus
growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no
c
-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an
E. coli plsC
(Ts) mutant of either
R. capsulatus plsC316
or
olsA
gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that
plsC316
encodes the main AGPAT required for glycerophospholipid synthesis in
R. capsulatus
, while
olsA
acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of
R. capsulatus
, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.
H2O2Is Required for Optimal Establishment of theMedicago sativa/Sinorhizobium melilotiSymbiosis
Authors: Jamet Alexandre , Mandon Karine , Puppo Alain , Hérouart Didier
Published: October 2007

The symbiotic interaction betweenMedicago sativaandSinorhizobium melilotiRmkatB++overexpressing the housekeeping catalasekatBis delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H2O2is required for optimal progression of infection threads through the root hairs and plant cell layers.
Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon
Archaeoglobus fulgidus
Strain 7324
Authors: Labes Antje , Schönheit Peter
Published: October 2007

The hyperthermophilic archaeon
Archaeoglobus fulgidus
strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, α-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that
A. fulgidus
utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly β-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from
Thermococcus litoralis
. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.
Structure, Function, and Regulation of the Aldouronate Utilization Gene Cluster from
Paenibacillus
sp. Strain JDR-2
Authors: Chow Virginia , Nong Guang , Preston James F.
Published: October 2007

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX
n
), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose.
Paenibacillus
sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX
n
and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX
3
, the aldotetrauronate 4-
O
-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as
xynA1
encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX
n
and repressed by glucose. Sequences in the
yesN, lplA
, and
xynA2
genes within the cluster and in the distal
xynA1
gene show significant similarity to catabolite responsive element (
cre
) defined in
Bacillus subtilis
for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in
Paenibacillus
sp. strain JDR-2 operates as a regulon, coregulated with the expression of
xynA1
, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.
TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology
Authors: César Carolina Elvira , Llosa Matxalen
Published: October 2007

R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate
oriT
s on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the
oriT
keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a “closed” conformation. In an IHF-deficient (IHF
−
) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF
−
background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short
oriT
loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an “open” form that exposes single-stranded DNA at the
nic
site, promoting the initial TrwC nicking reaction.
Orotate Phosphoribosyltransferase fromCorynebacterium ammoniagenesLacking a Conserved Lysine
Authors: Wang Xing , Ma Cuiqing , Wang Xiuwen , Xu Ping
Published: October 2007

ThepyrEgene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting fromCorynebacterium ammoniagenesATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 inSalmonella entericaserovar Typhimurium OPRTase) in theC. ammoniagenesOPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinantEscherichia colistrain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. TheKmvalues for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. TheKmvalue for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.
Effects of Oxygen on Virulence Traits ofStreptococcus mutans
Authors: Ahn Sang-Joon , Wen Zezhang T. , Burne Robert A.
Published: October 2007

Oxygen profoundly affects the composition of oral biofilms. Recently, we showed that exposure ofStreptococcus mutansto oxygen strongly inhibits biofilm formation and alters cell surface biogenesis. To begin to dissect the underlying mechanisms by which oxygen affects known virulence traits ofS. mutans, transcription profiling was used to show that roughly 5% of the genes of this organism are differentially expressed in response to aeration. Among the most profoundly upregulated genes were autolysis-related genes and those that encode bacteriocins, the ClpB protease chaperone subunit, pyruvate dehydrogenase, the tricarboxylic acid cycle enzymes, NADH oxidase enzymes, and certain carbohydrate transporters and catabolic pathways. Consistent with our observation that the ability ofS. mutansto form biofilms was severely impaired by oxygen exposure, transcription of thegtfBgene, which encodes one of the primary enzymes involved in the production of water-insoluble, adhesive glucan exopolysaccharides, was down-regulated in cells growing aerobically. Further investigation revealed that transcription ofgtfB, but notgtfC, was responsive to oxygen and that aeration causes major changes in the amount and degree of cell association of the Gtf enzymes. Moreover, inactivation of the VicK sensor kinase affected the expression and localization the GtfB and GtfC enzymes. This study provides novel insights into the complex transcriptional and posttranscriptional regulatory networks used byS. mutansto modulate virulence gene expression and exopolysaccharide production in response to changes in oxygen availability.
TheyydFGHIJOperon ofBacillus subtilisEncodes a Peptide That Induces the LiaRS Two-Component System
Authors: Butcher Bronwyn G. , Lin Yi-Pin , Helmann John D.
Published: October 2007

TheBacillus subtilisLiaRS two-component system (TCS) responds to perturbations of the cell envelope induced bylipid II-interactingantibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce theliaRSTCS. In addition to insertions inliaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of theyydFGHIJoperon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that theyydFGHIJoperon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent PliaIpromoter only in the presence of theyydFGHgenes. Cells harboring the completeyydFGHIJoperon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.