bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbazole-Degradative IncP-7 Plasmid pCAR1.2 Is Structurally Unstable in
Pseudomonas fluorescens
Pf0-1, Which Accumulates Catechol, the Intermediate of the Carbazole Degradation Pathway
Authors: Takahashi Yurika , Shintani Masaki , Li Li , Yamane Hisakazu , Nojiri Hideaki
Published: April 2009

We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazole-degradative plasmids pCAR1.1 and pCAR1.2. We constructed
Pseudomonas aeruginosa
PAO1(pCAR1.2) and
P. fluorescens
Pf0-1Km(pCAR1.2) and compared their growth on carbazole- and succinate-containing media with that of
P. putida
KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazole-degradative
car
operon was deleted or its regulatory gene,
antR
, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the
cat
genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.
Role of Ceftiofur in Selection and Dissemination of
bla
CMY-2
-Mediated Cephalosporin Resistance in
Salmonella enterica
and Commensal
Escherichia coli
Isolates from Cattle
Authors: Daniels Joshua B. , Call Douglas R. , Hancock Dale , Sischo William M. , Baker Katherine , Besser Thomas E.
Published: April 2009

Third-generation cephalosporin resistance of
Salmonella
and commensal
Escherichia coli
isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates β-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of
bla
CMY-2
-
-bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on
bla
CMY-2
transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent
bla
CMY-2
-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in
Salmonella
and commensal
E. coli
. The first study revealed
bla
CMY-2
plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (
P
= 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal
E. coli
. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible
Salmonella
isolates) used more ceftiofur than herds with no history of salmonellosis (
P
= 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of
bla
CMY-2
-bearing plasmid mediated cephalosporin resistance in commensal
E. coli
and in pathogenic
Salmonella
in these dairy cattle populations.
Effect of Amino Acid Availability on Vitamin B
12
Production in
Lactobacillus reuteri
Authors: Santos Filipe , Teusink Bas , Molenaar Douwe , van Heck Maurice , Wels Michiel , Sieuwerts Sander , de Vos Willem M. , Hugenholtz Jeroen
Published: April 2009

Recent functional genomics and genome-scale modeling approaches indicated that B
12
production in
Lactobacillus reuteri
could be improved by optimization of the medium. Here we show that a series of systematic single-amino-acid omissions could significantly modulate the production of B
12
from nearly undetectable levels (with omission of isoleucine) to levels 20-fold higher than the levels previously reported (with omission of cysteine). Using cDNA microarray experiments, we analyzed the transcriptional response of
L. reuteri
to medium lacking cysteine. The results supported the observed high level of B
12
production and provided new avenues for future improvement of production of vitamin B
12
.
Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in
Streptomyces virginiae
IBL-14
Authors: Wang Wei , Wang Feng-Qing , Wei Dong-Zhi
Published: April 2009

A new cytochrome P450 monooxygenase, FcpC, from
Streptomyces virginiae
IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an
Escherichia coli
strain.
CpxRA Contributes to
Xenorhabdus nematophila
Virulence through Regulation of
lrhA
and Modulation of Insect Immunity
Authors: Herbert Tran Erin E. , Goodrich-Blair Heidi
Published: April 2009

The gammaproteobacterium
Xenorhabdus nematophila
is a blood pathogen of insects that requires the CpxRA signal transduction system for full virulence (E. E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836, 2007). We show here that the Δ
cpxR1
mutant has altered localization, growth, and immune suppressive activities relative to its wild-type parent during infection of
Manduca sexta
insects. In contrast to wild-type
X. nematophila
, which were recovered throughout infection, Δ
cpxR1
cells did not accumulate in hemolymph until after insect death. In vivo imaging of fluorescently labeled bacteria within live insects showed that Δ
cpxR1
displayed delayed accumulation and also occasionally were present in isolated nodes rather than systemically throughout the insect as was wild-type
X. nematophila
. In addition, in contrast to its wild-type parent, the Δ
cpxR1
mutant elicited transcription of an insect antimicrobial peptide, cecropin. Relative to phosphate-buffered saline-injected insects, cecropin transcript was induced 21-fold more in insects injected with Δ
cpxR1
and 2-fold more in insects injected with wild-type
X. nematophila
. These data suggest that the Δ
cpxR1
mutant has a defect in immune suppression or has an increased propensity to activate
M. sexta
immunity. CpxR regulates, directly or indirectly, genes known or predicted to be involved in virulence (E. E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836, 2007), including
lrhA
, encoding a transcription factor necessary for
X. nematophila
virulence, motility, and lipase production (G. R. Richards et al., J. Bacteriol. 190:4870-4879, 2008). CpxR positively regulates
lrhA
transcript, and we have shown that altered regulation of
lrhA
in the Δ
cpxR1
mutant causes this strain's virulence defect. The Δ
cpxR1
mutant expressing
lrhA
from a constitutive
lac
promoter showed wild-type virulence in
M. sexta
. These data suggest that CpxR contributes to
X. nematophila
virulence through the regulation of
lrhA
, immune suppression, and growth in
Insecta
.
Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of
Vibrio parahaemolyticus
Isolates by Using Capillary Electrophoresis
Authors: Harth-Chu Erika , Espejo Romilio T. , Christen Richard , Guzmán Carlos A. , Höfle Manfred G.
Published: April 2009

Epidemics of
Vibrio parahaemolyticus
in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different
V. parahaemolyticus
isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental
V. parahaemolyticus
isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of
V. parahaemolyticus
belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of
V. parahaemolyticus
that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of
V. parahaemolyticus
with pandemic potential and provides a new surveillance tool for food-borne pathogens.
Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of
Burkholderia
spp. and Characterization of the
Burkholderia thailandensis betBA
Operon
Authors: Kang Yun , Norris Michael H. , Barrett Ashley R. , Wilcox Bruce A. , Hoang Tung T.
Published: April 2009

There are few appropriate single-copy genetic tools for most
Burkholderia
species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents
Burkholderia pseudomallei
and
Burkholderia mallei
, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel
r
) and
Escherichia coli
aspartate-semialdehyde dehydrogenase (

asd
Ec

), were developed to facilitate genetic manipulation in
Burkholderia
species. These systems include one mariner transposon, a mini-Tn
7
-derived site-specific transposon, and six
FRT
reporter fusion vectors based on the
lacZ
,
gfp
, and
luxCDABE
reporter genes. Initially, we showed that the random mariner transposon pBT20-Δ
bla
-Tel
r
-
FRT
efficiently transposed within
Burkholderia cenocepacia
,
Burkholderia thailandensis
,
B. pseudomallei
, and
B. mallei
. We then utilized the mini-Tn
7
-Tel
r
-based transposon vector (mini-Tn7-Tel
r
-
betBA
) and a transposase-containing helper plasmid (pTNS3-

asd
Ec

) to complement the
B. thailandensis
Δ
betBA
mutation. Next, one of the
FRT
-
lacZ
fusion vectors (pFRT1-
lacZ
-Tel
r
) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-
oriT
-

asd
Ec

) to construct the
B. thailandensis
Δ
betBA
::
FRT
-
lacZ
-Tel
r
reporter fusion strain. The
betBA
operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the
B. thailandensis
Δ
betBA
::
FRT
-
lacZ
-Tel
r
fusion strain. Finally, we engineered
B. thailandensis
Δ
betBA
::
FRT
-
gfp
-Tel
r
and Δ
betBA
::
FRT
-
lux
-Tel
r
fusion strains by utilizing fusion vectors pFRT1-
gfp
-Tel
r
and pFRT1-
lux
-Tel
r
, respectively. The induction of the
betBA
operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.
Tracking Functional Guilds: “Dehalococcoides” spp. in European River Basins Contaminated with Hexachlorobenzene
Authors: Taş Neslihan , van Eekert Miriam H. A. , Schraa Gosse , Zhou Jizhong , de Vos Willem M. , Smidt Hauke
Published: April 2009

Hexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. “Dehalococcoides” spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of activeDehalococcoidesspp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase (rdh) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance ofDehalococcoidesspp. but also in the composition of the populations andrdhgene diversity. Multivariate statistics revealed thatDehalococcoidessp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of activeDehalococcoidesspp. in HCB-contaminated river basins.
Increased Antibiotic Resistance ofEscherichia coliin Mature Biofilms
Authors: Ito Akinobu , Taniuchi Asami , May Thithiwat , Kawata Koji , Okabe Satoshi
Published: April 2009

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility ofEscherichia colicells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced byrpoSexpression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction ofrpoS-mediated stress responses.
Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of
Entamoeba histolytica
, Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin
Authors: Welter B. H. , Temesvari L. A.
Published: April 2009

Entamoeba histolytica
is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence.
E. histolytica
possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/
N
-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in
E. histolytica
.
Transducin Beta-Like Gene
FTL1
Is Essential for Pathogenesis in
Fusarium graminearum
Authors: Ding Shengli , Mehrabi Rahim , Koten Cornelia , Kang Zhensheng , Wei Yangdou , Seong Kyeyong , Kistler H. Corby , Xu Jin-Rong
Published: April 2009

Fusarium head blight caused by
Fusarium graminearum
is an important disease of wheat and barley. In a previous study, we identified several mutants with reduced virulence by insertional mutagenesis. A transducin beta-like gene named
FTL1
was disrupted in one of these nonpathogenic mutants.
FTL1
is homologous to
Saccharomyces cerevisiae SIF2
, which is a component of the Set3 complex involved in late stages of ascospore formation. The Δ
ftl1
mutant was significantly reduced in conidiation and failed to cause typical disease symptoms. It failed to colonize the vascular tissues of rachis or cause necrosis on the rachis of inoculated wheat heads. The Δ
ftl1
mutant also was defective in spreading from infected anthers to ovaries and more sensitive than the wild type to plant defensins MsDef1 and osmotin. However, the activation of two mitogen-activated protein kinases, Mgv1 and Gpmk1, production of deoxynivalenol, and expression of genes known to be important for plant infection in
F. graminearum
were not affected, indicating that the defect of the Δ
ftl1
mutant in plant infection is unrelated to known virulence factors in this pathogen and may involve novel mechanisms. The Δ
ftl1
deletion mutant was significantly reduced in histone deacetylation, and many members of the yeast Set3 complex are conserved in
F. graminearum
.
FTL1
appears to be a component of this well-conserved protein complex that plays a critical role in the penetration and colonization of wheat tissues.
Flavin Mononucleotide-Based Fluorescent Protein as an Oxygen-Independent Reporter in
Candida albicans
and
Saccharomyces cerevisiae
Authors: Tielker D. , Eichhof I. , Jaeger K.-E. , Ernst J. F.
Published: April 2009

Hypoxia is encountered frequently by pathogenic and apathogenic fungi. A codon-adapted gene encoding flavin mononucleotide-based fluorescent protein (Ca
FbFP
) was expressed in
Candida albicans
and
Saccharomyces cerevisiae
. Both species produced CaFbFP and fluoresced even during hypoxia, suggesting that oxygen-independent CaFbFP is a useful, novel tool for monitoring hypoxic gene expression in fungi.
Development of amariner-Based Transposon and Identification ofListeria monocytogenesDeterminants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype
Authors: Zemansky Jason , Kline Benjamin C. , Woodward Joshua J. , Leber Jess H. , Marquis Hélène , Portnoy Daniel A.
Published: April 2009

Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogenListeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed aHimar1 marinertransposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar toyjbH), lmo1268 (clpX), lmo1401 (similar toymdB), lmo1575 (similar toytqI), lmo1695 (mprF), lmo1821 (similar toprpC), lmo2219 (prsA2), and lmo2460 (similar tocggR). Some of these genes are involved in previously unexplored areas of research withL. monocytogenes: the genesyjbHandclpXregulate the disulfide stress response inBacillus subtilis, and theprpCphosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate thatprsA2, an extracytoplasmic peptidyl-prolylcis/transisomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown thatprsA2expression is linked to PrfA, the master virulence transcription factor inL. monocytogenespathogenesis, we demonstrate thatprsA2is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.
α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in
Pseudomonas fluorescens
Authors: Mailloux Ryan J. , Singh Ranji , Brewer Guy , Auger Christopher , Lemire Joseph , Appanna Vasu D.
Published: April 2009

α-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon,
Pseudomonas fluorescens
was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH
4
) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media.
13
C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of
P. fluorescens
and also point to the ROS-sensing role for KGDH.
The
Escherichia coli
Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage
Authors: Polosina Yaroslava Y. , Mui Justin , Pitsikas Photini , Cupples Claire G.
Published: April 2009

The activities of the Vsr and MutH endonucleases of
Escherichia coli
are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the
mutY
gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.
The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N
α
Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of
Haloferax volcanii
Authors: Humbard Matthew A. , Zhou Guangyin , Maupin-Furlow Julie A.
Published: April 2009

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N
α
acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of
Haloferax volcanii
proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in
H. volcanii
cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N
α
acetylation. Both the “gate” deletion and Q2A substitution completely altered the N
α
-acetylation pattern of α1, with the deletion rendering α1 unavailable for N
α
acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N
α
acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N
α
-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N
α
acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.
A Metabolic Operon in Extraintestinal PathogenicEscherichia coliPromotes Fitness under Stressful Conditions and Invasion of Eukaryotic Cells
Authors: Rouquet Géraldine , Porcheron Gaëlle , Barra Claire , Répérant Maryline , Chanteloup Nathalie K. , Schouler Catherine , Gilot Philippe
Published: April 2009

We identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenicEscherichia coli(ExPEC) strains. Thefrzoperon codes for three subunits of aphosphoenolpyruvate:carbohydrate phosphotransferasesystem (PTS) transporter of the fructose subfamily, for a transcriptional activator of PTSs of the MgA family, for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase (repressor, open reading frame, kinase family [ROK]), and for a protein of the cupin superfamily. We proved that thefrzoperon promotes bacterial fitness under stressful conditions, such as oxygen restriction, late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed thatfrzis involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of thefimoperon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PTS activator and the metabolic enzymes encoded by thefrzoperon are involved in these phenotypes.
Citrate Utilization byCorynebacterium glutamicumIs Controlled by the CitAB Two-Component System through Positive Regulation of the Citrate Transport GenescitHandtctCBA
Authors: Brocker Melanie , Schaffer Steffen , Mack Christina , Bott Michael
Published: April 2009

In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacteriumCorynebacterium glutamicumwas studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded bycitHbelongs to the citrate-Mg2+:H+/citrate-Ca2+:H+symporter family, whereas the permease encoded by thetctCBAoperon is a member of the tripartite tricarboxylate transporter family. The expression ofcitHortctCBAinEscherichia colienabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinantE. colistrains indicated that CitH is active with Ca2+or Sr2+but not with Mg2+and that TctABC is active with Ca2+or Mg2+but not with Sr2+. We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, theC. glutamicumwild type grew best when the minimal medium was supplemented with CaCl2but that MgCl2and SrCl2also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression ofcitHandtctCBAwas activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates.C. glutamicumCitAB is the first member of this family studied inActinobacteria.
Genomic Sequencing Reveals Regulatory Mutations and Recombinational Events in the Widely Used MC4100 Lineage ofEscherichia coliK-12
Authors: Ferenci Thomas , Zhou Zhemin , Betteridge Thu , Ren Yan , Liu Yu , Feng Lu , Reeves Peter R. , Wang Lei
Published: April 2009

The genome of anEscherichia coliMC4100 strain with a λ placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.
Conserved Network of Proteins Essential for Bacterial Viability
Authors: Handford Jennifer I. , Ize Bérengère , Buchanan Grant , Butland Gareth P. , Greenblatt Jack , Emili Andrew , Palmer Tracy
Published: April 2009

TheyjeE,yeaZ, andygjDgenes are highly conserved in the genomes of eubacteria, andygjDorthologs are also found throughout theArchaeaand eukaryotes. In this study, we have constructed conditional expression strains for each of these genes in the model organismEscherichia coliK12. We show that each gene is essential for the viability ofE. coliunder laboratory growth conditions. Growth of the conditional strains under nonpermissive conditions results in dramatic changes in cell ultrastructure. Deliberate repression of the expression ofyeaZresults in cells with highly condensed nucleoids, while repression ofyjeEandygjDexpression results in at least a proportion of very enlarged cells with an unusual peripheral distribution of DNA. Each of the three conditional expression strains can be complemented by multicopy clones harboring therstAgene, which encodes a two-component-system response regulator, strongly suggesting that these proteins are involved in the same essential cellular pathway. The results of bacterial two-hybrid experiments show that YeaZ can interact with both YjeE and YgjD but that YgjD is the preferred interaction partner. The results of in vitro experiments indicate that YeaZ mediates the proteolysis of YgjD, suggesting that YeaZ and YjeE act as regulators to control the activity of this protein. Our results are consistent with these proteins forming a link between DNA metabolism and cell division.
Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic
Escherichia coli
CFT073
Authors: Totsika Makrina , Heras Begoña , Wurpel Daniël J. , Schembri Mark A.
Published: April 2009

D
i
s
ulfide
b
ond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic
Escherichia coli
(UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in
trans
on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic
dsbAB
deletion mutant of CFT073 was severely attenuated, while deletion of
dsbLI
or
assT
did not affect colonization.
Quorum-Sensing Control of Antibiotic Synthesis in
Burkholderia thailandensis
Authors: Duerkop Breck A. , Varga John , Chandler Josephine R. , Peterson Snow Brook , Herman Jake P. , Churchill Mair E. A. , Parsek Matthew R. , Nierman William C. , Greenberg E. Peter
Published: April 2009

The genome of
Burkholderia thailandensis
codes for several LuxR-LuxI quorum-sensing systems. We used
B. thailandensis
quorum-sensing deletion mutants and recombinant
Escherichia coli
to determine the nature of the signals produced by one of the systems, BtaR2-BtaI2, and to show that this system controls genes required for the synthesis of an antibiotic. BtaI2 is an acyl-homoserine lactone (acyl-HSL) synthase that produces two hydroxylated acyl-HSLs,
N
-3-hydroxy-decanoyl-HSL (3OHC
10
-HSL) and
N
-3-hydroxy-octanoyl-HSL (3OHC
8
-HSL). The
btaI2
gene is positively regulated by BtaR2 in response to either 3OHC
10
-HSL or 3OHC
8
-HSL. The
btaR2-btaI2
genes are located within clusters of genes with annotations that suggest they are involved in the synthesis of polyketide or peptide antibiotics. Stationary-phase cultures of wild-type
B. thailandensis
, but not a
btaR2
mutant or a strain deficient in acyl-HSL synthesis, produced an antibiotic effective against gram-positive bacteria. Two of the putative antibiotic synthesis gene clusters require BtaR2 and either 3OHC
10
-HSL or 3OHC
8
-HSL for activation. This represents another example where antibiotic synthesis is controlled by quorum sensing, and it has implications for the evolutionary divergence of
B. thailandensis
and its close relatives
Burkholderia pseudomallei
and
Burkholderia mallei
.
Inhibition of Transcription inStaphylococcus aureusby a Primary Sigma Factor-Binding Polypeptide from Phage G1
Authors: Dehbi Mohammed , Moeck Gregory , Arhin Francis F. , Bauda Pascale , Bergeron Dominique , Kwan Tony , Liu Jing , McCarty John , DuBow Michael , Pelletier Jerry
Published: April 2009

The primary sigma factor ofStaphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSAboth in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSAthat is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSAto DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed inS. aureuscells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed inEscherichia coliconcurs with its inability to inhibit transcription by theE. coliholoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 forS. aureusRNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.
The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase
Authors: Rao Feng , Qi Yaning , Chong Hui Shan , Kotaka Masayo , Li Bin , Li Jinming , Lescar Julien , Tang Kai , Liang Zhao-Xun
Published: April 2009

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α)8barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 fromPseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (Km) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease inKmcorrelates with a change of solvent accessibility in the loop 6 region. We further examinedAcetobacter xylinusdiguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg2+ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.
A Unique Group I Intron in
Coxiella burnetii
Is a Natural Splice Mutant
Authors: Raghavan Rahul , Hicks Linda D. , Minnick Michael F.
Published: April 2009

Cbu.L1917, a group I intron present in the 23S rRNA gene of
Coxiella burnetii
, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.
The
N
-Acetylmannosamine Transferase Catalyzes the First Committed Step of Teichoic Acid Assembly in
Bacillus subtilis
and
Staphylococcus aureus
Authors: D'Elia Michael A. , Henderson James A. , Beveridge Terry J. , Heinrichs David E. , Brown Eric D.
Published: April 2009

There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the
N
-acetylmannosamine transferase (
tagA
in
Bacillus subtilis
;
tarA
in
Staphylococcus aureus
), which adds
N
-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the
N
-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that
tagA
(or
tarA
) encodes the first committed step in wall teichoic acid synthesis.
Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites
Authors: Zampini Massimiliano , Derome Andrew , Bailey Simon E. S. , Barillà Daniela , Hayes Finbarr
Published: April 2009

The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of theparFGpartition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel β-strands in this fold are presumed to insert into the OFoperator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The OFlocus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. OFwas subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A ≈ B ≫ D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The OFoperator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.
Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple
Staphylococcus aureus
Genomes Identify Novel Antimicrobial Drug Targets
Authors: Lee Deok-Sun , Burd Henry , Liu Jiangxia , Almaas Eivind , Wiest Olaf , Barabási Albert-László , Oltvai Zoltán N. , Kapatral Vinayak
Published: April 2009

Mortality due to multidrug-resistant
Staphylococcus aureus
infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for
S. aureus
infections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant
S. aureus
strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant
S. aureus
strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13
S. aureus
strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in
S. aureus
. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.
Mutations in Flk, FlgG, FlhA, and FlhE That Affect the Flagellar Type III Secretion Specificity Switch in
Salmonella enterica
Authors: Hirano Takanori , Mizuno Shino , Aizawa Shin-Ichi , Hughes Kelly T.
Published: April 2009

Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in
flk
-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (
flgG
* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev.
21:
2326-2335, 2007). A fusion of β-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance (Ap
r
). Ap
r
resulted from null mutations in the
flhE
gene, C-terminal truncation mutations in the
flhA
gene, null and dominant mutations in the
flk
gene, and
flgG
* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (FlgJ) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the
flgG
* and
flk
mutant strains, but it was in the
flhE
mutants. Unexpectedly, when deletions of either
flgE
or
flgD
were introduced into
flgG
* mutant strains, filaments were able to grow directly on the filamentous rod structures.
Functional Characterization of Excision Repair and RecA-Dependent Recombinational DNA Repair in
Campylobacter jejuni
Authors: Gaasbeek Esther J. , van der Wal Fimme J. , van Putten Jos P. M. , de Boer Paulo , van der Graaf-van Bloois Linda , de Boer Albert G. , Vermaning Bart J. , Wagenaar Jaap A.
Published: April 2009

The presence and functionality of DNA repair mechanisms in
Campylobacter jejuni
are largely unknown. In silico analysis of the complete translated genome of
C. jejuni
NCTC 11168 suggests the presence of genes involved in methyl-directed mismatch repair (MMR), nucleotide excision repair, base excision repair (BER), and recombinational repair. To assess the functionality of these putative repair mechanisms in
C. jejuni
,
mutS
,
uvrB
,
ung
, and
recA
knockout mutants were constructed and analyzed for their ability to repair spontaneous point mutations, UV irradiation-induced DNA damage, and nicked DNA. Inactivation of the different putative DNA repair genes did not alter the spontaneous mutation frequency. Disruption of the UvrB and RecA orthologues, but not the putative MutS or Ung proteins, resulted in a significant reduction in viability after exposure to UV irradiation. Assays performed with uracil-containing plasmid DNA showed that the putative uracil-DNA glycosylase (Ung) protein, important for initiation of the BER pathway, is also functional in
C. jejuni
. Inactivation of
recA
also resulted in a loss of natural transformation. Overall, the data indicate that
C. jejuni
has multiple functional DNA repair systems that may protect against DNA damage and limit the generation of genetic diversity. On the other hand, the apparent absence of a functional MMR pathway may enhance the frequency of on-and-off switching of phase variable genes typical for
C. jejuni
and may contribute to the genetic heterogeneity of the
C. jejuni
population.