bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Indigoidine Biosynthetic Genes in
Erwinia chrysanthemi
and Role of This Blue Pigment in Pathogenicity
Authors: Reverchon Sylvie , Rouanet Carine , Expert Dominique , Nasser William
Published: April 2009

In the plant-pathogenic bacterium
Erwinia chrysanthemi
production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain,
E. chrysanthemi ind
mutants deficient in indigoidine synthesis were isolated by screening a library of Tn
5
-B21 insertions in a
pecS
mutant. These
ind
mutations were localized close to the regulatory
pecS-pecM
locus, immediately downstream of
pecM
. Sequence analysis of this DNA region revealed three open reading frames,
indA
,
indB
, and
indC
, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of
ind
genes is strongly derepressed in the
pecS
background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses
indA
and
indC
expression by binding to
indA
and
indC
promoter regions. The regulatory link, via
pecS
, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in
E. chrysanthemi
pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted
Saintpaulia ionantha
. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.
Nested Evolution of a tRNA
Leu
(UAA) Group I Intron by both Horizontal Intron Transfer and Recombination of the Entire tRNA Locus
Authors: Rudi Knut , Fossheim Tonje , Jakobsen Kjetill S.
Published: April 2009

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA
Leu
(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA
Ile
(CAU) and tRNA
Arg
(CCU) genes in α- and β-proteobacteria, respectively, than with other cyanobacterial tRNA
Leu
(UAA) group I introns. The divergent tRNA
Leu
(UAA) intron is sporadically distributed both within the
Nostoc
and the
Microcystis
radiations. The complete tRNA gene, including flanking regions and intron from
Microcystis aeruginosa
strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the
Microcystis
genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA
Leu
(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA
Leu
(UAA) gene which is located within a unstable genome region.
Novel Pathway for Alcoholic Fermentation of δ-Gluconolactone in the YeastSaccharomyces bulderi
Authors: van Dijken Johannes P. , van Tuijl Arjen , Luttik Marijke A. H. , Middelhoven Wouter J. , Pronk Jack T.
Published: April 2009

Under anaerobic conditions, the yeastSaccharomyces bulderirapidly ferments δ-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for δ-gluconolactone fermentation operates in this yeast. In this pathway, δ-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in δ-gluconolactone fermentation inS. bulderiis supported by several experimental observations. (i) Fermentation of δ-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as δ-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP+-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, δ-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on δ-gluconolactone, CO2production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO2. (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in δ-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in δ-gluconolactone metabolism.
Analysis of
ftsQ
Mutant Alleles in
Escherichia coli
: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins
Authors: Chen Joseph C. , Minev Michael , Beckwith Jon
Published: April 2009

FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium
Escherichia coli
. To define residues in FtsQ critical for function, we performed random mutagenesis on the
ftsQ
gene and identified four alleles (
ftsQ2
,
ftsQ6
,
ftsQ15
, and
ftsQ65
) that fail to complement the
ftsQ1
(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an
ftsQ1
(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.
High-Salinity-Induced Iron Limitation in
Bacillus subtilis
Authors: Hoffmann Tamara , Schütz Alexandra , Brosius Margot , Völker Andrea , Völker Uwe , Bremer Erhard
Published: April 2009

Proteome analysis of
Bacillus subtilis
cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the
dhbACEBF
operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed
dhb
expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed
B. subtilis
cultures. Taken together, these findings strongly suggest that
B. subtilis
cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of
B. subtilis
in natural settings, in which bioavailable iron is usually scarce.
Regulation of
mutY
and Nature of Mutator Mutations in
Escherichia coli
Populations under Nutrient Limitation
Authors: Notley-McRobb Lucinda , Pinto Rachel , Seeto Shona , Ferenci Thomas
Published: April 2009

Previous analysis of aerobic, glucose-limited continuous cultures of
Escherichia coli
revealed that G:C-to-T:A (G:C→T:A) transversions were the most commonly occurring type of spontaneous mutation. One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:C→T:A transversions. The regulation of the
mutY
-dependent DNA repair system was therefore studied with a transcriptional
mutY-lacZ
fusion recombined into the chromosome. Expression from the
mutY
promoter was fourfold higher under aerobic conditions than under anaerobic conditions. But
mutY
expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so
mutY
was not downregulated by nutrient limitation. An alternative explanation for the frequency of G:C→T:A transversions was the common appearance of
mutY
mutator mutations in the chemostat populations. Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of
mutY
at 66 min on the chromosome. The spectrum of mutations and rate of mutation in these isolates were fully consistent with a
mutY
-deficiency in each strain. Based on PCR analysis of the region within and around
mutY
, isolates from three individual populations contained deletions extending at least 2 kb upstream of
mutY
and more than 5 kb downstream. In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of
mutY
. The isolation of
mutY
mutator strains from four independent populations with extensive chromosomal rearrangements suggests that
mutY
inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:C→T:A mutations in glucose-limited chemostats.
Rubrivivax gelatinosus acsF
(Previously
orf358
) Codes for a Conserved, Putative Binuclear-Iron-Cluster-Containing Protein Involved in Aerobic Oxidative Cyclization of Mg-Protoporphyrin IX Monomethylester
Authors: Pinta Violaine , Picaud Martine , Reiss-Husson Françoise , Astier Chantal
Published: April 2009

This study describes the characterization of
orf358
, an open reading frame of previously unidentified function, in the purple bacterium
Rubrivivax gelatinosus
. A strain in which
orf358
was disrupted exhibited a phenotype similar to the wild type under photosynthesis or low-aeration respiratory growth conditions. In contrast, under highly aerated respiratory growth conditions, the wild type still produced bacteriochlorophyll
a
(Bchl
a
), while the disrupted strain accumulated a compound that had the same absorption and fluorescence emission spectra as Mg-protoporphyrin but was less polar, suggesting that it was Mg-protoporphyrin monomethylester (MgPMe). These data indicated a blockage in Bchl
a
synthesis at the oxidative cyclization stage and implied the coexistence of two different mechanisms for MgPMe cyclization in
R. gelatinosus
, an anaerobic mechanism active under photosynthesis or low oxygenation and an aerobic mechanism active under high-oxygenation growth conditions. Based on these results as well as on sequence analysis indicating the presence of conserved putative binuclear-iron-cluster binding motifs, the designation of
orf358
as
acsF
(for aerobic cyclization system Fe-containing subunit) is proposed. Several homologs of AcsF were found in a wide range of photosynthetic organisms, including
Chlamydonomas reinhardtii
Crd1 and
Pharbitis nil
PNZIP, suggesting that this aerobic oxidative cyclization mechanism is conserved from bacteria to plants.
Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain
Authors: Rashid Naeem , Cornista Joel , Ezaki Satoshi , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: April 2009

A gene encoding a cyclodextrin glucanotransferase (CGTase) from
Thermococcus kodakaraensis
KOD1 (CGT

Tk

) was identified and characterized. The gene (
cgt

Tk

) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the α-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca
2+
enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca
2+
, the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT

Tk

could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT

Tk

harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with β-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT

Tk

ΔC) with 23 C-terminal amino acid residues deleted. CGT

Tk

ΔC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT

Tk

. In contrast, the cyclization activity of CGT

Tk

ΔC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT

Tk

to properly catalyze the cyclization reaction.
Galactose and Lactose Genes from the Galactose-Positive Bacterium
Streptococcus salivarius
and the Phylogenetically Related Galactose-Negative Bacterium
Streptococcus thermophilus
: Organization, Sequence, Transcription, and Activity of the
gal
Gene Products
Authors: Vaillancourt Katy , Moineau Sylvain , Frenette Michel , Lessard Christian , Vadeboncoeur Christian
Published: April 2009

Streptococcus salivarius
is a lactose- and galactose-positive bacterium that is phylogenetically closely related to
Streptococcus thermophilus
, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the
S. salivarius
and
S. thermophilus gal-lac
gene clusters. The clusters have the same organization with the order
galR
(codes for a transcriptional regulator and is transcribed in the opposite direction),
galK
(galactokinase),
galT
(galactose-1-P uridylyltransferase),
galE
(UDP-glucose 4-epimerase),
galM
(galactose mutarotase),
lacS
(lactose transporter), and
lacZ
(β-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from
galR
, the
gal
operon,
galM
, and the
lac
operon of
S. salivarius
. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the
S. thermophilus gal
-
lac
gene cluster. An additional putative internal promoter at the 3′ end of
galT
was found in
S. thermophilus
but not in
S. salivarius
. The results clearly indicated that the
gal-lac
gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of
galT
and
galE
were identical in both species, whereas the ribosome binding site of
S. thermophilus galK
differed from that of
S. salivarius
by two nucleotides, suggesting that the
S. thermophilus galK
gene might be poorly translated. This was confirmed by measurements of enzyme activities.
Regulation of the
Streptomyces coelicolor
Calcium-Dependent Antibiotic by
absA
, Encoding a Cluster-Linked Two-Component System
Authors: Ryding N. Jamie , Anderson Todd B. , Champness Wendy C.
Published: April 2009

The
Streptomyces coelicolor absA
two-component system was initially identified through analysis of mutations in the sensor kinase
absA1
that caused inhibition of all four antibiotics synthesized by this strain. Previous genetic analysis had suggested that the phosphorylated form of AbsA2 acted as a negative regulator of antibiotic biosynthesis in
S. coelicolor
(T. B. Anderson, P. Brian, and W. C. Champness, Mol. Microbiol. 39:553–566, 2001). Genomic sequence data subsequently provided by the Sanger Centre (Cambridge, United Kingdom) revealed that
absA
was located within the gene cluster for production of one of the four antibiotics, calcium-dependent antibiotic (CDA). In this paper we have identified numerous transcriptional start sites within the CDA cluster and have shown that the original antibiotic-negative mutants used to identify
absA
exhibit a stronger negative regulation of promoters upstream of the proposed CDA biosynthetic genes than of promoters in the clusters responsible for production of actinorhodin and undecylprodigiosin. The same antibiotic-negative mutants also showed an increase in transcription from a promoter divergent to that of
absA
, upstream of a putative ABC transporter, in addition to an increase in transcription of
absA
itself. Interestingly, the negative regulation of the biosynthetic transcripts did not appear to be mediated by transcriptional regulation of
cdaR
(a gene encoding a homolog of the pathway-specific regulators of the
act
and
red
clusters) or by any other recognizable transcriptional regulator associated with the cluster. The role of
absA
in regulating the expression of the diverse antibiotic biosynthesis clusters in the genome is discussed in light of its location in the
cda
cluster.
Role of Menaquinones in Fe(III) Reduction by Membrane Fractions of
Shewanella putrefaciens
Authors: Saffarini Daad A. , Blumerman Seth L. , Mansoorabadi Karen J.
Published: April 2009

Two Tn
5
-generated mutants of
Shewanella putrefaciens
with insertions in
menD
and
menB
were isolated and analyzed. Both mutants were deficient in the use of several terminal electron acceptors, including Fe(III). This deficiency was overcome by the addition of menaquinone (vitamin K
2
). Isolated membrane fractions from both mutants were unable to reduce Fe(III) in the absence of added menaquinone when formate was used as the electron donor. These results indicate that menaquinones are essential components for the reduction of Fe(III) by both whole cells and purified membrane fractions when formate or lactate is used as the electron donor.
Evidence for a Fourth Hydrogenase in
Desulfovibrio fructosovorans
Authors: Casalot Laurence , De Luca Gilles , Dermoun Zorah , Rousset Marc , de Philip Pascale
Published: April 2009

A strain devoid of the three hydrogenases characterized for
Desulfovibrio fructosovorans
was constructed using marker exchange mutagenesis. As expected, the H
2
-dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H
+
-D
2
exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in
D. fructosovorans
.
Integrative Genetic Element That Reverses the Usual Target Gene Orientation
Authors: Zhao Sihui , Williams Kelly P.
Published: April 2009

A genetic element integrating site specifically into a prokaryotic gene usually carries a copy of the 3′ portion of that gene that restores the active gene even as the original is disrupted. A cryptic element in
Mesorhizobium loti
instead carries a copy of the 5′ end of the tRNA gene into which it integrated. This has implications for the evolution of new integrase-site combinations.
Replication-Dependent Recruitment of the β-Subunit of DNA Polymerase III from Cytosolic Spaces to Replication Forks in
Escherichia coli
Authors: Onogi Toshinari , Ohsumi Katsufumi , Katayama Tsutomu , Hiraga Sota
Published: April 2009

The β-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoid-occupied space in exponentially growing cells of
Escherichia coli
. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive
dnaC
mutant, the β-subunit is located in the cytosolic spaces of the cell poles.
Corynebacterium glutamicumUtilizes both Transsulfuration and Direct Sulfhydrylation Pathways for Methionine Biosynthesis
Authors: Hwang Byung-Joon , Yeom Hye-Jin , Kim Younhee , Lee Heung-Shick
Published: April 2009

A direct sulfhydrylation pathway for methionine biosynthesis inCorynebacterium glutamicumwas found. The pathway was catalyzed bymetYencodingO-acetylhomoserine sulfhydrylase. The genemetY, located immediately upstream ofmetA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction ofmetYintoC. glutamicumresulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase inO-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of themetBgene, which encodes cystathionine γ-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or themetYgene was not enough to lead to methionine auxotrophy, an additional mutation in themetYor themetBgene resulted in methionine auxotrophy. The growth pattern of themetYmutant strain was identical to that of themetBmutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, anEscherichia coli metBmutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterialmetYandmetAgenes. These data clearly show thatC. glutamicumutilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. AlthoughmetYandmetAare in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As withmetA, methionine could also repress the expression ofmetY. The repression was also observed withmetB, but the degree of repression was more severe withmetY,which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway inC. glutamicum.
YopD and LcrH Regulate Expression of
Yersinia enterocolitica
YopQ by a Posttranscriptional Mechanism and Bind to
yopQ
RNA
Authors: Anderson Deborah M. , Ramamurthi Kumaran S. , Tam Christina , Schneewind Olaf
Published: April 2009

Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 μM. To characterize the mechanism that regulates the expression of
yopQ
, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in
yopN
,
tyeA
,
sycN
, or
yscB
synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in
yopD
or
lcrH
synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that
yopD
and
lcrH
are required for the regulation of
yopQ
expression. Experiments with transcriptional and translational fusions to the
npt
reporter gene suggest that
yopD
and
lcrH
regulate
yopQ
expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind
yopQ
mRNA. Models that can account for posttranscriptional regulatory mechanisms of
yop
expression are discussed.
Amino Acid- and Purine Ribonucleoside-Induced Germination of
Bacillus anthracis
ΔSterne Endospores:
gerS
Mediates Responses to Aromatic Ring Structures
Authors: Ireland John A. W. , Hanna Philip C.
Published: April 2009

Specific combinations of amino acids or purine ribonucleosides and amino acids are required for efficient germination of endospores of
Bacillus anthracis
ΔSterne, a plasmidless strain, at ligand concentrations in the low-micromolar range. The amino acid
l
-alanine was the only independent germinant in
B. anthracis
and then only at concentrations of >10 mM. Inosine and
l
-alanine both play major roles as cogerminants with several other amino acids acting as efficient cogerminants (His, Pro, Trp, and Tyr combining with
l
-alanine and Ala, Cys, His, Met, Phe, Pro, Ser, Trp, Tyr, and Val combining with inosine). An ortholog to the
B. subtilis
tricistronic germination receptor operon
gerA
was located on the
B. anthracis
chromosome and named
gerS
. Disruption of
gerS
completely eliminated the ability of
B. anthracis
endospores to respond to amino-acid and inosine-dependent germination responses. The
gerS
mutation also produced a significant microlag in the aromatic-amino-acid-enhanced-alanine germination pathways. The
gerS
disruption appeared to specifically affect use of aromatic chemicals as cogerminants with alanine and inosine. We conclude that efficient germination of
B. anthracis
endospores requires multipartite signals and that
gerS
-encoded proteins act as an aromatic-responsive germination receptor.
Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration byEscherichia coliK-12
Authors: Stewart Valley , Lu Yiran , Darwin Andrew J.
Published: April 2009

Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especiallyParacoccus pantotrophus. Whole-genome sequencing ofEscherichia colirevealed the structural genesnapFDAGHBC, which encode NapABC enzyme and associated electron transfer components.E. colialso expresses two membrane-bound proton-translocating nitrate reductases, encoded by thenarGHJIandnarZYWVoperons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations ofnarandnapnull alleles. ThenapFoperon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for theP. pantotrophusNapA enzyme. A strain carrying null alleles ofnarGandnarZgrew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele ofnapAdid not. By contrast, the presence ofnapA+had no influence on the more rapid growth ofnarG+strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function inE. coliwhen low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.
YplA Is Exported by the Ysc, Ysa, and Flagellar Type III Secretion Systems ofYersinia enterocolitica
Authors: Young Briana M. , Young Glenn M.
Published: April 2009

Yersinia enterocoliticamaintains three different pathways for type III protein secretion. Each pathway requires the activity of a specific multicomponent apparatus or type III secretion system (TTSS). Two of the TTSSs are categorized as contact-dependent systems which have been shown in a number of different symbiotic and pathogenic bacteria to influence interactions with host organisms by targeting effector proteins into the cytosol of eukaryotic cells. The third TTSS is required for the assembly of flagella and the secretion of the phospholipase YplA, which has been implicated inY. enterocoliticavirulence. In this study, YplA was expressed from a constitutive promoter in strains that contained only a single TTSS. It was determined that each of the three TTSSs is individually sufficient for YplA secretion. Environmental factors such as temperature, calcium availability, and sodium chloride concentration affected the contribution of each system to extracellular protein secretion and, under some conditions, more than one TTSS appeared to operate simultaneously. This suggests that some proteins might normally be exported by more than one TTSS inY. enterocolitca.
Two Novel Type III-Secreted Proteins of
Xanthomonas campestris
pv. vesicatoria Are Encoded within the
hrp
Pathogenicity Island
Authors: Noël Laurent , Thieme Frank , Nennstiel Dirk , Bonas Ulla
Published: April 2009

The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen
Xanthomonas campestris
pv. vesicatoria. cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key
hrp
regulator HrpG, in the regions flanking the
hrp
gene cluster. Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeat-containing protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from
Xanthomonas oryzae
pv. oryzae and PsvA from
Pseudomonas syringae
, respectively. XopA and XopD (
Xanthomonas
outer proteins) are secreted by the
Xanthomonas
Hrp TTSS and thus represent putative effector proteins. Mutations in
xopA
, but not in
xopD
, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants. Since the
xopD
promoter contains a putative
hrp
box, which is characteristic of
hrpL
-regulated genes in
P. syringae
and
Erwinia
spp., the gene was probably acquired by horizontal gene transfer. Interestingly, the regions flanking the
hrp
gene cluster also contain insertion sequences and genes for a putative transposase and a tRNA
Arg
. These features suggest that the
hrp
gene cluster of
X. campestris
pv. vesicatoria is part of a pathogenicity island.
rRNA Promoter Activity in the Fast-Growing Bacterium
Vibrio natriegens
Authors: Aiyar Sarah E. , Gaal Tamas , Gourse Richard L.
Published: April 2009

The bacterium
Vibrio natriegens
can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that
V. natriegens
' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that
V. natriegens
contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The
V. natriegens
rRNA core promoters are at least as active in vitro as
Escherichia coli
rRNA core promoters with either
E. coli
RNA polymerase (RNAP) or
V. natriegens
RNAP, and they are activated by UP elements, as in
E. coli
. In addition, the
E. coli
transcription factor Fis activated
V. natriegens rrn
P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in
V. natriegens
results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in
E. coli
, i.e., high gene dose and strong activation by UP elements and Fis.
Cel9M, a New Family 9 Cellulase of the
Clostridium cellulolyticum
Cellulosome
Authors: Belaich Anne , Parsiegla Goetz , Gal Laurent , Villard Claude , Haser Richard , Belaich Jean-Pierre
Published: April 2009

A new cellulosomal protein from
Clostridium cellulolyticum
Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from
C. cellulolyticum
, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.
In Vitro Serial Passage of
Staphylococcus aureus
: Changes in Physiology, Virulence Factor Production, and
agr
Nucleotide Sequence
Authors: Somerville Greg A. , Beres Stephen B. , Fitzgerald J. Ross , DeLeo Frank R. , Cole Robert L. , Hoff Jessica S. , Musser James M.
Published: April 2009

Recently, we observed that
Staphylococcus aureus
strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (
P
< 0.001) by the sixth week in culture. During serial passage,
S. aureus
existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (
agr
) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (ρ = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the
agrC
coding region or the intergenic region between
agrC
and
agrA
in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the
agr
operon. Additionally, our results demonstrate that
S. aureus
undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.
Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of
Helicobacter pylori
Authors: Wolfram Lutz , Bauerfeind Peter
Published: April 2009

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen
Helicobacter pylori
. The nickel permease NixA of
H. pylori
is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in
Escherichia coli
, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79—both part of the characteristic sequence motif—and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the
E. coli
system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.
Charged Amino Acids Conserved in the Aromatic Acid/H
+
Symporter Family of Permeases Are Required for 4-Hydroxybenzoate Transport by PcaK from
Pseudomonas putida
Authors: Ditty Jayna L. , Harwood Caroline S.
Published: April 2009

Charged amino acids in the predicted transmembrane portion of PcaK, a permease from
Pseudomonas putida
that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for
P
.
putida
to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H
+
symporter family of the major facilitator superfamily of transporters.
Glucose-Resistant Sporulation in
Bacillus subtilis crsA47
Mutants Does Not Depend on Promoter Switching at the
spo0A
Gene
Authors: Dixon Laurie G. , Spiegelman George B.
Published: April 2009

We have found that sporulation in
Bacillus subtilis crsA47
mutants does not require the σ
H
-dependent promoter of the
spo0A
gene. This implies that the glucose-resistant sporulation phenotype of this strain is not related to the switch from the vegetative-stage σ
A
-dependent promoter to the σ
H
-dependent promoter at the
spo0A
gene.
New Class of IMP Cyclohydrolases in
Methanococcus jannaschii
Authors: Graupner Marion , Xu Huimin , White Robert H.
Published: April 2009

The enzyme responsible for observed IMP cyclohydrolase activity in
Methanococcus jannaschii
was purified and sequenced: its genetic locus was found to correspond to gene MJ0626. The MJ0626 gene was cloned, and its protein product was expressed in
Escherichia coli
and shown to catalyze the cyclization of 5-formylamidoimidazole-4-carboxamide ribonucleotide to IMP. The enzyme has no sequence similarity to known enzymes, and its catalytic properties appear distinct from any characterized IMP cyclohydrolase. The
purO
gene for the enzyme is currently found only in the domain
Archaea
.
AmfS, an Extracellular Peptidic Morphogen in
Streptomyces griseus
Authors: Ueda Kenji , Oinuma Ken-Ichi , Ikeda Go , Hosono Kuniaki , Ohnishi Yasuo , Horinouchi Sueharu , Beppu Teruhiko
Published: April 2009

The
amf
gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in
Streptomyces griseus
. The nucleotide sequences of
amf
and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in
amfS
, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the
amfS
mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in
amfBA
, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the
amfS
mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.
Transcription of the SsrAB Regulon Is Repressed by Alkaline pH and Is Independent of PhoPQ and Magnesium Concentration
Authors: Miao Edward A. , Freeman Jeremy A. , Miller Samuel I.
Published: April 2009

TheSalmonellapathogenicity island 2 (SPI-2) type III secretion system is expressed by intracellular bacteria and translocates effector proteins across the vacuolar membrane. Signals sensed bySalmonella entericaserovar Typhimurium in the intracellular compartment activate SPI-2 gene expression through the two-component regulatory system SsrAB. The effects of environmental and genetic signals on expression of the SsrAB-regulated genesspH2were examined. SsrAB-dependent activation ofsspH2was detected in the presence of both low and moderate concentrations of magnesium or calcium and at acidic and neutral pHs. The levels of expression were comparable to those detected in bacteria recovered from cultured macrophages. The induction in media at alkaline pHs (pH 7.5 and 8.0) was greatly reduced compared to the induction observed at pH 7.0 or at a lower pH, suggesting that alkaline pH represses SsrAB activation. In addition, the PhoPQ two-component system, which is also activated intracellularly, was not required for activation of SsrAB.
Characterization of the
petI
and
res
Operons of
Acidithiobacillus ferrooxidans
Authors: Levicán Gloria , Bruscella Patrice , Guacunano Maritza , Inostroza Carolina , Bonnefoy Violaine , Holmes David S. , Jedlicki Eugenia
Published: April 2009

DNA sequence analysis and bioinformatic interpretations have identified two adjacent clusters of genes potentially involved in the formation of a bc1 complex and in the maturation of a cytochrome
c
-type protein in two strains (ATCC 19859 and ATCC 33020) of the acidophilic, chemolithoautotrophic bacterium
Acidithiobacillus ferrooxidans
(formerly
Thiobacillus ferrooxidans
). Reverse transcriptase-PCR experiments suggest that the two clusters are organized as operons, and +1 start sites of transcription for the operons have been determined by primer extension experiments. Potential promoters have been identified. The presence of these operons lends support to a recent model of reverse electron flow and is consistent with previous reports of phenotypic switching in this bacterium.
Spectrum of Zygomycete Species Identified in Clinically Significant Specimens in the United States
Authors: Alvarez E. , Sutton D. A. , Cano J. , Fothergill A. W. , Stchigel A. , Rinaldi M. G. , Guarro J.
Published: April 2009

Several members of the order
Mucorales
(subphylum
Mucoromycotina
) are important agents of severe human infections. The identification of these fungi by using standard mycologic methods is often difficult and time consuming. Frequently, the etiological agent in clinical cases is reported either as a
Mucor
sp., which is not the most frequent genus of zygomycetes, or only as a member of the
Mucorales
. For this reason, the actual spectrum of species of zygomycetes and their incidences in the clinical setting is not well known. The goals of this study were to compare the results of the molecular identification of an important set of clinical isolates, received in a mycological reference center from different regions of the United States, with those obtained by using the traditional morphological methods and to determine the spectrum of species involved. We tested 190 isolates morphologically identified as zygomycetes by using sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. Molecular identification revealed that
Rhizopus oryzae
represented approximately half (44.7%) of these isolates. The remainder was identified as
Rhizopus microsporus
(22.1%),
Mucor circinelloides
(9.5%),
Mycocladus corymbifer
(formerly
Absidia corymbifera
) (5.3%),
Rhizomucor pusillus
(3.7%),
Cunninghamella bertholletiae
(3.2%),
Mucor indicus
(2.6%),
Cunninghamella echinulata
(1%), and
Apophysomyces elegans
(0.5%). The most common anatomic sites for clinically significant zygomycetes, as determined by isolates sent to the Fungus Testing Laboratory for identification and/or susceptibility testing and included in this study, were the sinuses, lungs, and various cutaneous locations, at 25.8%, 26.8%, and 28%, respectively. These sites represented approximately 80% of the isolates evaluated. A high level of correlation (92.6%) between morphological and molecular identifications was found.
First Spanish Case of Nocardiosis Caused by
Nocardia takedensis
Authors: Betrán A. , Rezusta A. , Lezcano M. A. , Villuendas M. C. , Revillo M. J. , Boiron P. , Rodríguez-Nava V.
Published: April 2009

Nocardia takedensis
is a recently described species isolated from soil. The first clinical isolate in Japan has recently been reported. This report describes the first clinical isolate of
N. takedensis
in Spain from a respiratory specimen.
Fine-Needle Aspiration, an Efficient Sampling Technique for Bacteriological Diagnosis of Nonulcerative Buruli Ulcer
Authors: Eddyani Miriam , Fraga Alexandra G. , Schmitt Fernando , Uwizeye Cécile , Fissette Krista , Johnson Christian , Aguiar Julia , Sopoh Ghislain , Barogui Yves , Meyers Wayne M. , Pedrosa Jorge , Portaels Françoise
Published: April 2009

Invasive punch or incisional skin biopsy specimens are currently employed for the bacteriological confirmation of the clinical diagnosis of Buruli ulcer (BU), a cutaneous infectious disease caused byMycobacterium ulcerans. The efficacy of fine-needle aspirates (FNA) using fine-gauge needles (23G by 25 mm) for the laboratory confirmation of BU was compared with that of skin tissue fragments obtained in parallel by excision or punch biopsy. In three BU treatment centers in Benin, both types of diagnostic material were obtained from 33 clinically suspected cases of BU and subjected to the same laboratory analyses: i.e., direct smear examination, IS2404PCR, and in vitro culture. Twenty-three patients, demonstrating 17 ulcerative and 6 nonulcerative lesions, were positive by at least two tests and were therefore confirmed to have active BU. A total of 68 aspirates and 68 parallel tissue specimens were available from these confirmed patients. When comparing the sensitivities of the three confirmation tests between FNA and tissue specimens, the latter yielded more positive results, but only for PCR was this significant. When only nonulcerative BU lesions were considered, however, the sensitivities of the confirmation tests using FNA and tissue specimens were not significantly different. Our results show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in nonulcerative lesions.