bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chitinase B of “
Microbulbifer degradans
” 2-40 Contains Two Catalytic Domains with Different Chitinolytic Activities
Authors: Howard Michael B. , Ekborg Nathan A. , Taylor Larry E. , Weiner Ronald M. , Hutcheson Steven W. 
Published: February 2004

ABSTRACT

Chitinase B of “
Microbulbifer degradans
” 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18
N
) releases methylumbelliferone from 4′-methylumbelliferyl-
N
,
N
′-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18
C
) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18
C
releases methylumbelliferone from 4′-methylumbelliferyl-
N
,
N
′,
N
"-triacetylchitotriose 2.7-fold faster than GH18
N
and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37°C and from pH 7.2 to 8.0 and was not affected by Mg
2+
, Mn
2+
, Ca
2+
, K
+
, EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni
2+
, Sr
2+
, and Cu
2+
, while Hg
2+
completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.
In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination
Authors: Torres Martha , Balada Joan-Miquel , Zellars Malcolm , Squires Craig , Squires Catherine L. 
Published: February 2004

ABSTRACT

Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rho-dependent terminator. A comparison of terminator read-through in a wild-type
Escherichia coli
strain and that in a
nusB
::IS
10
mutant strain determined the requirement for NusB. In the absence of NusB, antiterminator-dependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusG-depleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.
Identification and Mapping of Self-Assembling Protein Domains Encoded by theEscherichia coliK-12 Genome by Use of λ Repressor Fusions
Authors: Mariño-Ramírez Leonardo , Minor Jonathan L. , Reading Nicola , Hu James C. 
Published: February 2004

ABSTRACTSelf-assembling proteins and protein fragments encoded by theEscherichia coligenome were identified fromE. coliK-12 strain MG1655. Libraries of random DNA fragments cloned into a series of λ repressor fusion vectors were subjected to selection for immunity to infection by phage λ. Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232E. coligenes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online athttp://oligomers.tamu.edu/.
Functionally Critical Elements of CooA-Related CO Sensors
Authors: Youn Hwan , Kerby Robert L. , Conrad Mary , Roberts Gary P. 
Published: February 2004

ABSTRACT

CooA is a heme-containing transcriptional activator that enables
Rhodospirillum rubrum
to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in
Escherichia coli
, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from
Azotobacter vinelandii
and
Carboxydothermus hydrogenoformans
corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of
R. rubrum
. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.
Dimerization of the RamC Morphogenetic Protein of
Streptomyces coelicolor
Authors: Hudson Michael E. , Nodwell Justin R. 
Published: February 2004

ABSTRACT

RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium
Streptomyces coelicolor
. This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanism of action. In this study we found that the presence of multiple copies of a defective allele of
ramC
inhibits morphogenesis in
S. coelicolor
, consistent with either titration of a target or formation of inactive RamC multimers. We identified a domain in RamC that is C terminal to the putative kinase domain and forms a dimer with a

K
d

of ∼0.1 μM. These data suggest that RamC acts as a dimer in vivo.
The
Pseudomonas putida
Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds
Authors: Morales Gracia , Linares Juan Francisco , Beloso Ana , Albar Juan Pablo , Martínez José Luis , Rojo Fernando 
Published: February 2004

ABSTRACT

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in
Pseudomonas putida
and
Pseudomonas aeruginosa
when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic
P. putida
strains containing either a wild-type or an inactivated
crc
allele was compared. The results showed that Crc is involved in the catabolic repression of the
hpd
and
hmgA
genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in
P. putida
. It was found that genes required to assimilate benzoate through the catechol pathway (
benA
and
catBCA
) and 4-OH-benzoate through the protocatechuate pathway (
pobA
and
pcaHG
) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in
P. putida
.
In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of
Streptomyces noursei
ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis
Authors: Sekurova Olga N. , Brautaset Trygve , Sletta Håvard , Borgos Sven E. F. , Jakobsen Øyvind M. , Ellingsen Trond E. , Strøm Arne R. , Valla Svein , Zotchev Sergey B. 
Published: February 2004

ABSTRACT

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in
Streptomyces noursei
ATCC 11455. Gene inactivation and complementation experiments revealed that
nysRI
,
nysRII
,
nysRIII
, and
nysRIV
are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the
xylE
reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the
S. noursei
wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that
nysR
genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.
Expression of Cholera Toxin under Non-AKI Conditions in
Vibrio cholerae
El Tor Induced by Increasing the Exposed Surface of Cultures
Authors: Sánchez Joaquín , Medina Gerardo , Buhse Thomas , Holmgren Jan , Soberón-Chavez Gloria 
Published: February 2004

ABSTRACT

The regulatory systems controlling expression of the
ctxAB
genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic
Vibrio cholerae
have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.
In Silico and Transcriptional Analysis of Carbohydrate Uptake Systems of
Streptomyces coelicolor
A3(2)
Authors: Bertram Ralph , Schlicht Maximilian , Mahr Kerstin , Nothaft Harald , Saier Milton H. , Titgemeyer Fritz 
Published: February 2004

ABSTRACT

Streptomyces coelicolor
is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of
S. coelicolor
A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, α-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and β-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and
N
-acetylglucosamine, the genome of
S. coelicolor
encodes at least 53 potential carbohydrate uptake systems.
Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in
Xanthomonas oryzae
pv. oryzae
Authors: Furutani Ayako , Tsuge Seiji , Ohnishi Kouhei , Hikichi Yasufumi , Oku Takashi , Tsuno Kazunori , Inoue Yasuhiro , Ochiai Hirokazu , Kaku Hisatoshi , Kubo Yasuyuki 
Published: February 2004

ABSTRACT

Xanthomonas oryzae
pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient
hrp
-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of
X. oryzae
pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the
hrp
regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by
hrp
genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that
cysP2
has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a
cysP2
::
gus
fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the
hrp
regulatory gene
hrpXo
is also involved in the expression of not only
hrp
genes and type III secretory proteins but also some type II secretory proteins.
Studies of the Interaction of
Escherichia coli
YjeQ with the Ribosome In Vitro
Authors: Daigle Denis M. , Brown Eric D. 
Published: February 2004

ABSTRACT

Escherichia coli
YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of
E. coli
and
Bacillus subtilis
. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h
−1
) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry
41:
11109-11117, 2002). In the work reported here, YjeQ from
E. coli
was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5′-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.
Transcriptional Organization and Regulation of the
l
-Idonic Acid Pathway (GntII System) in
Escherichia coli
Authors: Bausch Christoph , Ramsey Matthew , Conway Tyrrell 
Published: February 2004

ABSTRACT

The genetic organization of the
idn
genes that encode the pathway for
l
-idonate catabolism was characterized. The monocistronic
idnK
gene is transcribed divergently from the
idnDOTR
genes, which were shown to form an operon. The 215-bp regulatory region between the
idnK
and
idnD
genes contains promoters in opposite orientation with transcription start sites that mapped to positions −26 and −29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of
idnD
, and an UP element upstream of
idnK
. The genes of the
l
-idonate pathway were shown to be under catabolite repression control. Analysis of
idnD
- and
idnK
-
lacZ
fusions in a nonpolar
idnD
mutant that is unable to interconvert
l
-idonate and 5-ketogluconate indicated that either compound could induce the pathway. The
l
-idonate pathway was first characterized as a subsidiary pathway for
d
-gluconate catabolism (GntII), which is induced by
d
-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the
idnK
and
idnD
operons are induced by
d
-gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from
d
-gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in
l
-idonate catabolism; the GntII system can be induced by
d
-gluconate under conditions that block the GntI system.
Relevance of Peptide Uptake Systems to the Physiology and Virulence of
Streptococcus agalactiae
Authors: Samen Ulrike , Gottschalk Birgit , Eikmanns Bernhard J. , Reinscheid Dieter J. 
Published: February 2004

ABSTRACT

Streptococcus agalactiae
is a major cause of invasive infections in human newborns. To satisfy its growth requirements,
S. agalactiae
takes up 9 of the 20 proteinogenic amino acids from the environment. Defined
S. agalactiae
mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by
S. agalactiae
was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes
oppA1
-
F
and
dppA
-
E
and a monocistronic organization of
dpsA
in
S. agalactiae
. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons
dppA
-
E
and
oppA1
-
F
in
S. agalactiae
. Growth of
S. agalactiae
in human amniotic fluid was shown to require an intact
dpsA
gene, indicating an important role of DpsA during the infection of the amniotic cavity by
S. agalactiae
. Deletion of the
oppB
gene reduced the adherence of
S. agalactiae
to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the
fbsA
gene, which encodes a fibrinogen-binding protein in
S. agalactiae
. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in
S. agalactiae
.
A Dominant-NegativefurMutation inBradyrhizobium japonicum
Authors: Benson Heather P. , LeVier Kristin , Guerinot Mary Lou 
Published: February 2004

ABSTRACTIn many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiontBradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified afurmutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of thefurgene repress iron uptake systems and starve for iron, whereasfurmutants fail to repress iron uptake systems and survive. TheB. japonicum furmutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of thefurgene cannot complement thefurmutant. Expression of thefurmutant allele in wild-type cells leads to afurphenotype. Unlike aB. japonicum fur-null mutant, the strain carrying the dominant-negativefurmutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.