bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of Genes Required for Early
Myxococcus xanthus
Developmental Gene Expression
Authors: Guo Dongchuan , Wu Yun , Kaplan Heidi B. 
Published: July 2002

ABSTRACT

Starvation and cell density regulate the developmental expression of
Myxococcus xanthus
gene
4521
. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn
5 lac
Ω4521 fusion are Lac
+
. One class of these mutants inactivates SasN, a negative regulator of
4521
expression; another class activates SasS, a sensor kinase-positive regulator of
4521
expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of
4521
expression, 11 Lac
−
Tn
V.AS
transposon insertion mutants were isolated from a screen of 18,000 Lac
+
LPS O-antigen mutants containing Tn
5 lac
Ω4521 (Tc
r
). Ten mutations identified genes that could encode positive regulators of
4521
developmental expression based on their ability to abolish
4521
expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the
sasB
locus, which encodes the known
4521
regulators SasS and SasN. One mapped to
sasS
, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated
sasR
, and four others mapped to a gene designated
sasP
. One mutation, designated
ssp10
, specifically suppressed the LPS O-antigen defect; the
ssp10
mutation had no effect on
4521
expression in an otherwise wild-type background but reduced
4521
developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in
sasP
conferred defects during growth and development. These data indicate that a number of elements are required for
4521
developmental expression and that most of these are necessary for normal growth and fruiting body development.
Effects of the Calvin Cycle on Nicotinamide Adenine Dinucleotide Concentrations and Redox Balances of
Xanthobacter flavus
Authors: van Keulen Geertje , Dijkhuizen Lubbert , Meijer Wim G. 
Published: July 2002

ABSTRACT

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in
Xanthobacter flavus
during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.
Biochemical and Molecular Characterization of Phenylacetate-Coenzyme A Ligase, an Enzyme Catalyzing the First Step in Aerobic Metabolism of Phenylacetic Acid in
Azoarcus evansii
Authors: El-Said Mohamed Magdy 
Published: July 2002

ABSTRACT

Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC
6.2.1.30
), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in
Azoarcus evansii
, has been purified and characterized. The gene (
paaK
) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min
−1
mg of cell protein
−1
was measured. After 117-fold purification to homogeneity, a specific activity of 48 μmol min
−1
mg of protein
−1
was achieved with a turnover number (catalytic constant) of 40 s
−1
. The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent

K
m

values for PA, ATP, and CoA were 14, 60, and 45 μM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1,320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium.
Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively AlkaliphilicBacillus pseudofirmusOF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily
Authors: Gilmour Raymond , Messner Paul , Guffanti Arthur A. , Kent Rebecca , Scheberl Andrea , Kendrick Nancy , Krulwich Terry Ann 
Published: July 2002

ABSTRACTThe large majority of proteins of alkaliphilicBacillus pseudofirmusOF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well.slpARNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. AnslpAmutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na+-replete or suboptimal Na+concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na+. By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na+-replete and suboptimal Na+conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na+-dependent transport of α-aminoisobutyric acid, or H+-dependent synthesis of ATP. However, the capacity for Na+-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.
BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in
Pseudomonas putida
Authors: Cowles Charles E. , Nichols Nancy N. , Harwood Caroline S. 
Published: July 2002

ABSTRACT

Pseudomonas putida
converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded
meta
pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of
P. putida
to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of
pcaK
, the gene encoding the 4-HBA permease. Here we identified a gene,
benR
, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A
benR
mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to
benR
were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The
benABC
genes likely encode benzoate dioxygenase, and
benD
likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase.
benK
and
benF
were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene,
benE
, is not known.
benR
activated expression of a
benA-lacZ
reporter fusion in response to benzoate. It also activated expression of a
meta
cleavage operon promoter-
lacZ
fusion inserted in an
E. coli
chromosome. Third,
benR
was required for benzoate-mediated repression of
pcaK-lacZ
fusion expression. The
benA
promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the
meta
cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.
The Malonate Decarboxylase Operon of
Acinetobacter calcoaceticus
KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY
Authors: Koo Jae Hyung , Cho Ick Hyun , Kim Yu Sam 
Published: July 2002

ABSTRACT

A regulatory gene-like open reading frame oriented oppositely to mdcL, coined
mdcY
, was found upstream from the structural genes of the
mdcLMACDEGBH
operon in
Acinetobacter calcoaceticus
KCCM 40902. To elucidate the function of this gene,
mdcY
was expressed in
Escherichia coli
, and the MdcY protein was purified to homogeneity. Its DNA binding activity and binding site were examined by gel retardation and footprinting assays in vitro and by site-directed mutagenesis of the binding sites in vivo. The regulator bound target DNA regardless of the presence of malonate, and the binding site was found centered at −65 relative to the
mdcL
transcriptional start site and contains a 12-bp palindromic structure (5′-ATTGTA/TACAAT-3′). Using a promoter fusion to the reporter gene
luc
, we found that the promoter P

mdcY

is negatively regulated by MdcY independent of malonate. However, the promoter P

mdcL

recovered its activity in the presence of malonate. When
mdcY
was introduced into
A. calcoaceticus
KCCM 40902 in which the gene is inactivated by an IS
3
family element, malonate decarboxylase was significantly repressed in cultures growing in acetate, succinate, or Luria-Bertani medium. However, in cells growing in malonate, malonate decarboxylase was induced, indicating that MdcY is a transcriptional repressor and that malonate or a product resulting from malonate metabolism should be the intracellular inducer of the
mdc
operon.
Flavodoxin Mutants of
Escherichia coli
K-12
Authors: Gaudu Philippe , Weiss Bernard 
Published: July 2002

ABSTRACT

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of
Escherichia coli
, and its gene,
fldA
, is known to belong to the
soxRS
(superoxide response) oxidative stress regulon. An insertion mutation of
fldA
was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (
fldA
+
) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene,
fldB
. Unlike the
fldA
mutant, an
fldB
insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number
fldB
+
plasmid did not complement the
fldA
mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An
fldB-lacZ
fusion was not induced by H
2
O
2
and is therefore not a member of the
oxyR
regulon. However, it displayed a
soxS
-dependent induction by paraquat (methyl viologen), and the
fldB
gene is preceded by two overlapping regions that resemble known
soxS
binding sites. The
fldB
insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a
soxS-lacZ
fusion. Therefore,
fldB
is a new member of the
soxRS
(superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.
Osmoregulated Periplasmic Glucan Synthesis Is Required for
Erwinia chrysanthemi
Pathogenicity
Authors: Page Frederic , Altabe Silvia , Hugouvieux-Cotte-Pattat Nicole , Lacroix Jean-Marie , Robert-Baudouy Janine , Bohin Jean-Pierre 
Published: July 2002

ABSTRACT

Erwinia chrysanthemi
is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the
opgGH
operon of
E. chrysanthemi
, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus
mdoGH
of
Escherichia coli
. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the
opgG
or
opgH
gene abolish OPG synthesis. The
opg
mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.
Evidence that an Additional Mutation Is Required To Tolerate Insertional Inactivation of theStreptomyces lividans recAGene
Authors: Vierling Silke , Weber Tilmann , Wohlleben Wolfgang , Muth Günther 
Published: July 2002

ABSTRACTIn contrast torecAof other bacteria, therecAgene ofStreptomyces lividanshas been described as indispensable for viability (G. Muth, D. Frese, A. Kleber, and W. Wohlleben, Mol. Gen. Genet. 255:420–428, 1997.). Therefore, a closer analysis of this gene was performed to detect possible unique features distinguishing theStreptomycesRecA protein from the well-characterizedEscherichia coliRecA protein. TheS. lividans recAgene restored UV resistance and recombination activity of anE. coli recAmutant. Also, transcriptional regulation was similar to that ofE. coli recA. Gel retardation experiments showed thatS. lividans recAis also under control of theStreptomycesSOS repressor LexA. TheS. lividans recAgene could be replaced only by simultaneously expressing a plasmid encodedrecAcopy. Surprisingly, therecAexpression plasmid could subsequently be eliminated using an incompatible plasmid without the loss of viability. Besides being UV sensitive and recombination deficient, all the mutants were blocked in sporulation. Genetic complementation restored UV resistance and recombination activity but did not affect the sporulation defect. This indicated that all therecAmutants had suffered from an additional mutation, which might allow toleration of arecAdeficiency.
Respiration Capacity of the Fermenting Bacterium
Lactococcus lactis
and Its Positive Effects on Growth and Survival
Authors: Duwat Patrick , Sourice Sophie , Cesselin Bénédicte , Lamberet Gilles , Vido Karin , Gaudu Philippe , Le Loir Yves , Violet Florent , Loubière Pascal , Gruss Alexandra 
Published: July 2002

ABSTRACT

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe
Lactococcus lactis
, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to
L. lactis
if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of
L. lactis
to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The
cydA
gene, encoding cytochrome
d
oxidase, is required for respiration and plays a direct role in oxygen utilization.
cydA
expression is induced late in growth under respiration conditions. (ii) The
hemZ
gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in
L. lactis
and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.
Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process
Authors: Park Hee-Sung , Kim Hak-Sung 
Published: July 2002

ABSTRACT

The aminophenol (AP) catabolic operon in
Pseudomonas putida
HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol
meta
cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene,
nbzR
, and the following nine structural genes,
nbzJCaCbDGFEIH
, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other
meta
cleavage operons led us to suggest that the regulatory unit (
nbzR
) was derived from the MarR family and that the structural unit (
nbzJCaCbDGFEIH
) has evolved from the ancestral
meta
cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.
Oxygen- and Growth Rate-Dependent Regulation of
Escherichia coli
Fumarase (FumA, FumB, and FumC) Activity
Authors: Tseng Ching-Ping , Yu Chin-Chu , Lin Hsiao-Hsien , Chang Chi-Yen , Kuo Jong-Tar 
Published: July 2002

ABSTRACT

Escherichia coli
contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to
l
-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (
k
) was lowered from 1.2/h to 0.24/h at 1 and 21% O
2
, respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a
cya
mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (
k
= 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.
Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin
Authors: Dairi Tohru , Hamano Yoshimitsu , Kuzuyama Tomohisa , Itoh Nobuya , Furihata Kazuo , Seto Haruo 
Published: July 2002

ABSTRACTA gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned fromStreptomyces griseolosporeusstrain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627–1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed inStreptomyces lividanstogether with the GGDP synthase gene under the control of theermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid,ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced inEscherichia coliand purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.