bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of α-Methylacyl Coenzyme A Racemase in the Degradation of Methyl-Branched Alkanes by
Mycobacterium
sp. Strain P101
Authors: Sakai Yasuyoshi , Takahashi Hironori , Wakasa Yuori , Kotani Tetsuya , Yurimoto Hiroya , Miyachi Nobuya , Van Veldhoven Paul P. , Kato Nobuo 
Published: October 2004

ABSTRACT

A new isolate,
Mycobacterium
sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn
5
-derived mutants, we characterized 2 mutants defective in growth on pristane or
n
-hexadecane. A single copy of Tn
5
was found to be inserted into the coding region of
mcr
(α-methylacyl coenzyme A [α-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of
mls
(malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in
Escherichia coli
was confirmed to catalyze racemization of (
R
)-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 μmol · min
−1
· mg of protein
−1
. Real-time quantitative reverse transcriptase PCR analyses indicated that
mcr
gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used (
S
)-2-methylbutyric acid for growth but did not use the racemic compound, and growth on
n
-hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the (
R
) isomer, although the (
R
) isomer was not toxic during growth on
n
-hexadecane. Based on these results, Mcr is suggested to play a critical role in β-oxidation of methyl-branched alkanes in
Mycobacterium
. On the other hand, mutant H1 could not grow on
n
-hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through β-oxidation of pristane.
MorA Defines a New Class of Regulators Affecting Flagellar Development and Biofilm Formation in Diverse
Pseudomonas
Species
Authors: Choy Weng-Keong , Zhou Lian , Syn Chris Kiu-Choong , Zhang Lian-Hui , Swarup Sanjay 
Published: October 2004

ABSTRACT

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in
Pseudomonas putida
. MorA is conserved among diverse
Pseudomonas
species, and homologues are present in all
Pseudomonas
genomes sequenced thus far. In
P. putida
, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In
Pseudomonas aeruginosa
, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of
P. putida
, the motility of the
P. aeruginosa
mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of
Pseudomonas
species.
The
Escherichia coli
DjlA and CbpA Proteins Can Substitute for DnaJ in DnaK-Mediated Protein Disaggregation
Authors: Gur Eyal , Biran Dvora , Shechter Nelia , Genevaux Pierre , Georgopoulos Costa , Ron Eliora Z. 
Published: October 2004

ABSTRACT

The DnaJ (Hsp40) protein of
Escherichia coli
serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other
E. coli
proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in
dnaJ
mutants both CbpA and DjlA are essential for growth at temperatures above 37°C, their in vivo role is poorly understood. Here we show that in a
dnaJ
mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42°C.
ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis
Authors: Tiemann Bernd , Depping Reinhard , Gineikiene Egle , Kaliniene Laura , Nivinskas Rimas , Rüger Wolfgang 
Published: October 2004

ABSTRACT
Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.
The
N
-Acetyl-
d
-Glucosamine Kinase of
Escherichia coli
and Its Role in Murein Recycling
Authors: Uehara Tsuyoshi , Park James T. 
Published: October 2004

ABSTRACT

N
-Acetyl-
d
-glucosamine (GlcNAc) is a major component of bacterial cell wall murein and the lipopolysaccharide of the outer membrane. During growth, over 60% of the murein of the side wall is degraded, and the major products, GlcNAc-anhydro-
N
-acetylmuramyl peptides, are efficiently imported into the cytoplasm and cleaved to release GlcNAc, anhydro-
N
-acetylmuramic acid, murein tripeptide (
l
-Ala-
d
-Glu-
meso
-diaminopimelic acid), and
d
-alanine. Like murein tripeptide, GlcNAc is readily recycled, and this process was thought to involve phosphorylation, since GlcNAc-6-phosphate (GlcNAc-6-P) is efficiently used to synthesize murein or lipopolysaccharide or can be metabolized by glycolysis. Since the gene for GlcNAc kinase had not been identified, in this work we purified GlcNAc kinase (NagK) from
Escherichia coli
cell extracts and identified the gene by determining the N-terminal sequence of the purified kinase. A
nagK
deletion mutant lacked phosphorylated GlcNAc in its cytoplasm, and the cell extract of the mutant did not phosphorylate GlcNAc, indicating that NagK is the only GlcNAc kinase expressed in
E. coli
. Unexpectedly, GlcNAc did not accumulate in a
nagK nagEBACD
mutant, though both GlcNAc and GlcNAc-6-P accumulate in the
nagEBACD
mutant, suggesting the existence of an alternative pathway (presumably repressed by GlcNAc-6-P) that reutilizes GlcNAc without the involvement of NagK.
Role of
hha
and
ler
in Transcriptional Regulation of the
esp
Operon of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Sharma Vijay K. , Zuerner Richard L. 
Published: October 2004

ABSTRACT

The locus of enterocyte effacement (LEE), which includes five major operons (
LEE1
through
LEE4
and
tir
), enables enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 to produce attaching and effacing lesions on host cells. Expression of
LEE2
,
LEE3
, and
tir
is positively regulated by
ler
, a gene located in
LEE1
. Transcriptional regulation of the
esp
operon (
LEE4
), however, is not well defined. Transposon mutagenesis was used to identify transcriptional regulators of the
esp
operon by screening for mutants with increased β-galactosidase activity in an EHEC O157:H7 strain harboring an
esp
::
lac
transcriptional fusion. All mutants with significant increases in β-galactosidase activity had transposon insertions in
hha
(
hha
::Tn). Specific complementation of the
hha
::Tn mutation with a plasmid-encoded copy of
hha
reduced β-galactosidase activity to the level expressed in the parental
esp
::
lac
strain. Purified Hha, however, bound poorly to the
esp
promoter, suggesting that Hha might repress the transcription of a positive regulator of
esp
. Transposon mutagenesis of a Δ
hha esp
::
lac
strain expressing elevated levels of β-galactosidase resulted in
ler
mutants with reduced β-galactosidase activity. Purified Hha bound to the
ler
promoter with a higher affinity, and complementation of a Δ
hha
mutation in a Δ
hha ler
::
lac
strain repressed β-galactosidase activity to the level expressed in a
ler
::
lac
strain. A positive regulatory role of
ler
in
esp
expression was demonstrated by specific binding of Ler to the
esp
promoter, reduced expression of β-galactosidase in Δ
ler esp
::
lac
strains with and without
hha
, and severalfold-increased transcription of
ler
and
espA
in strains lacking
hha
. These results indicate that
hha
-mediated repression of
ler
causes reduced expression of the
esp
operon.
The BfeR Regulator Mediates Enterobactin-Inducible Expression of
Bordetella
Enterobactin Utilization Genes
Authors: Anderson Mark T. , Armstrong Sandra K. 
Published: October 2004

ABSTRACT

Utilization of the enterobactin siderophore by the respiratory pathogens
Bordetella pertussis
and
Bordetella bronchiseptica
is dependent on the BfeA outer membrane receptor. This study determined that production of BfeA was increased significantly in iron-starved bacteria upon supplementation of cultures with enterobactin. A 1.01-kb open reading frame, designated
bfeR
, encoding a predicted positive transcriptional regulator of the AraC family was identified upstream and divergently oriented from
bfeA
. In iron-depleted cultures containing enterobactin, a
Bordetella bfeR
mutant exhibited markedly decreased BfeA receptor production compared to that of the wild-type strain. Additionally,
B. pertussis
and
B. bronchiseptica bfeR
mutants exhibited impaired growth with ferric enterobactin as the sole source of iron, demonstrating that effective enterobactin utilization is
bfeR
dependent. Transcriptional analysis using
bfeA
-
lacZ
reporter fusions in wild-type strains demonstrated that
bfeA
transcription was stimulated in iron-depleted conditions in the presence of enterobactin, compared to modest expression levels in cultures lacking enterobactin. In contrast,
bfeA
transcription in
B. pertussis
and
B. bronchiseptica bfeR
mutants was completely unresponsive to the enterobactin inducer.
bfeA
transcriptional analyses of a
bfeA
mutant demonstrated that induction by enterobactin did not require BfeA receptor-mediated uptake of the siderophore. These studies establish that
bfeR
encodes an enterobactin-dependent positive regulator of
bfeA
transcription in these
Bordetella
species.
Characterization of Nutrient-Induced Dispersion in
Pseudomonas aeruginosa
PAO1 Biofilm
Authors: Sauer K. , Cullen M. C. , Rickard A. H. , Zeef L. A. H. , Davies D. G. , Gilbert P. 
Published: October 2004

ABSTRACT

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of
Pseudomonas aeruginosa
PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to ∼80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (
fliC
) and correspondingly decreased expression of pilus (
pilA
) genes in dispersed cells. Changes in gene expression associated with dispersion of
P. aeruginosa
biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of
P. aeruginosa
from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of
P. aeruginosa
correlates with a specific dispersal phenotype.
A Nonribosomal Peptide Synthetase with a Novel Domain Organization Is Essential for Siderophore Biosynthesis in
Vibrio anguillarum
Authors: Di Lorenzo Manuela , Poppelaars Sophie , Stork Michiel , Nagasawa Maho , Tolmasky Marcelo E. , Crosa Jorge H. 
Published: October 2004

ABSTRACT

Anguibactin, a siderophore produced by
Vibrio anguillarum
, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. We have identified a gene from the
V. anguillarum
plasmid pJM1 that encodes a 78-kDa NRPS protein termed AngM, which is essential in the biosynthesis of anguibactin. The predicted AngM amino acid sequence shows regions of homology to the consensus sequence for the peptidyl carrier protein (PCP) and the condensation (C) domains of NRPSs, and curiously, these two domains are not associated with an adenylation (A) domain. Substitution by alanine of the serine 215 in the PCP domain and of histidine 406 in the C domain of AngM results in an anguibactin-deficient phenotype, underscoring the importance of these two domains in the function of this protein. The mutations in
angM
that affected anguibactin production also resulted in a dramatic attenuation of the virulence of
V. anguillarum
775, highlighting the importance of this gene in the establishment of a septicemic infection in the vertebrate host. Transcription of the
angM
gene is initiated at an upstream transposase gene promoter that is repressed by the Fur protein in the presence of iron. Analysis of the sequence at this promoter showed that it overlaps the iron transport-biosynthesis promoter and operates in the opposite direction.
The NtcA-Dependent P
1
Promoter Is Utilized for
glnA
Expression in N
2
-Fixing Heterocysts of
Anabaena
sp. Strain PCC 7120
Authors: Valladares Ana , Muro-Pastor Alicia M. , Herrero Antonia , Flores Enrique 
Published: October 2004

ABSTRACT

Expression of the
glnA
gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N
2
-fixing cyanobacterium
Anabaena
sp. strain PCC 7120,
glnA
is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNA
I
originates from a canonical NtcA-dependent promoter (P
1
) and RNA
II
originates from a σ
70
-type promoter (P
2
), RNA
IV
is influenced by NtcA but the corresponding promoter (P
3
) does not have the structure of NtcA-activated promoters. Using RNA isolated from
Anabaena
filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNA
V
, which has previously been detected only in in vitro transcription assays and should originate from P
4
. However, in heterocysts, which are differentiated cells specialized in N
2
fixation, RNA
I
was the almost exclusive
glnA
transcript. Analysis of P

glnA

::
lacZ
fusions containing different fragments of the
glnA
upstream region confirmed that fragments carrying P
1
, P
2
, or P
3
and P
4
have the ability to promote transcription. Mutation of the NtcA-binding site in P
1
eliminated P
1
-directed transcription and allowed increased use of P
2
. The NtcA-binding site in the P
1
promoter and binding of NtcA to this site appear to be key factors in determining
glnA
gene expression in vegetative cells and heterocysts.
Temporal Analysis of
Coxiella burnetii
Morphological Differentiation
Authors: Coleman Sherry A. , Fischer Elizabeth R. , Howe Dale , Mead David J. , Heinzen Robert A. 
Published: October 2004

ABSTRACT

Coxiella burnetii
undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study
C. burnetii
morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of
scvA
, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of
C. burnetii
is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of
C. burnetii
developmental forms.
AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in
Pseudomonas aeruginosa
Authors: Robles-Price Antonette , Wong Thiang Yian , Sletta Håvard , Valla Svein , Schiller Neal L. 
Published: October 2004

ABSTRACT

Alginate, an exopolysaccharide produced by
Pseudomonas aeruginosa
, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene,
algX
, within the alginate biosynthetic gene cluster on the
P. aeruginosa
chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of
algX
in
P. aeruginosa
strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting
algX
Δ::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The
algX
Δ::Gm mutant was restored to the mucoid phenotype with wild-type
P. aeruginosa algX
provided on a plasmid. The
algX
Δ::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.
Characterization of EvgAS-YdeO-GadE Branched Regulatory Circuit Governing Glutamate-Dependent Acid Resistance inEscherichia coli
Authors: Ma Zhuo , Masuda Nobuhisa , Foster John W. 
Published: October 2004

ABSTRACTEscherichia coliprefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the fourE. colisystems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, γ-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncoveredevgASandydeOas potential regulators ofgadE, now known to encode the essential activator of thegadAandgadBCgenes. Examination ofevgAandydeOunder normal expression conditions revealed that their products do activategadEexpression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary forgadEexpression in stationary-phase cells grown in complex medium. The response regulator EvgA activatesgadEdirectly and indirectly via induction of the AraC-like regulatorydeO. Evidence obtained usinggadE-lacZoperon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream ofgadE, indicating they all act directly at thegadEpromoter. Since GadE controls the expression of numerous genes besidesgadAandgadBC, the relevance of these regulatory circuits extends beyond acid resistance.
Use of In Vivo Expression Technology To Identify Genes Important in Growth and Survival of
Pseudomonas fluorescens
Pf0-1 in Soil: Discovery of Expressed Sequences with Novel Genetic Organization
Authors: Silby Mark W. , Levy Stuart B. 
Published: October 2004

ABSTRACT

Studies were undertaken to determine the genetic needs for the survival of
Pseudomonas fluorescens
Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.