bluuebunny/crossref_metadata_2025_split huggingface dataset:
MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence
Authors: Chatterjee Jaidip , Miyamoto Carol M. , Zouzoulas Athina , Lang B. Franz , Skouris Nicolas , Meighen Edward A. 
Published: March 2003

SummaryThe induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum‐sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA‐binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1000‐fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10‐fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.
Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation
Authors: Blauwkamp Timothy A. , Ninfa Alexander J. 
Published: March 2003

Summary Escherichia coli
contains two PII‐like signal trans‐duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of
glnALG
,
glnK
and
nac
during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled
glnALG
expression in cells adapted to growth on ammonia, but was unable to limit
nac
and
glnK
expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit
glnALG
expression in cells adapted to growth on ammonia, but was required to limit expression of the
glnK
and
nac
promoters during nitrogen starvation. In the absence of GlnK, very high expression of the
glnK
and
nac
promoters occurred in nitrogen‐starved cells, and the cells did not reduce
glnK
and
nac
expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild‐type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild‐type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate
nac
expression; deletion of
nac
restored wild‐type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the
glnK promoter in cells lacking GlnK restored the wild‐type phenotypes.
A family of autocrine growth factors in Mycobacterium tuberculosis
Authors: Mukamolova Galina V. , Turapov Obolbek A. , Young Danielle I. , Kaprelyants Arseny S. , Kell Douglas B. , Young Michael 
Published: March 2003

Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra‐cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross‐species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity‐purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.
A high‐molecular‐weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non‐fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence
Authors: Ray Suvendra K. , Rajeshwari R. , Sharma Yogendra , Sonti Ramesh V. 
Published: March 2003

SummaryTransposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1265 amino acids that exhibits significant similarity to non‐fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti‐XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a β‐helix conformation‐like pertactin, a non‐fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non‐fimbrial adhesin‐like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.
Selection of plasmid molecules for conjugative transfer and replacement strand synthesis in the donor
Authors: Parker Christopher , Meyer Richard J. 
Published: March 2003

SummaryPlasmid selection and strand replacement synthesis in donor cells during conjugative transfer was examined by a procedure involving electroporation of test plasmid DNA, containing a base pair mismatch, into donor cells prior to mating. Multiple copies of the plasmid were transferred from a donor cell that allowed vegetative replication of the plasmid. Under conditions non‐permissive for vegetative replication, there were further rounds of transfer after a lag period. Strand replacement in the donor did not depend solely on the initiation mechanism for vegetative replication, indicating a conjugation‐specific mechanism was also available. The lag period between first and second rounds of transfer argues against the transfer of multiple copies into recipients by the spooling of copies generated on a master molecule by rolling‐circle replication.
Knotting dynamics during DNA replication
Authors: Olavarrieta L. , Martínez‐Robles M. L. , Hernández P. , Krimer D. B. , Schvartzman J. B. 
Published: March 2003

SummaryThe topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two‐dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.
Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator
Authors: Yoshida Takeshi , Cai Sheng jian , Inouye Masayori 
Published: March 2003

SummaryEnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re‐examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR‐P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein‐labelled OmpR (Fl‐OmpR) with EnvZc was determined to be 1.96 ± 0.28 µM. We demonstrated that OmpR‐P as well as OmpR inhibited the interaction of Fl‐OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 ± 0.25 µM and 0.89 ± 0.14 µM, respectively, under the conditions used. The interaction between His‐10‐OmpR and EnvZc was also inhibited almost equally with OmpR‐P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR‐P (phosphatase), indicating that EnvZc is able to interact with Fl‐OmpR or Fl‐OmpR‐P as well as with OmpR or OmpR‐P as a substrate. We demonstrated that OmpR‐P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR‐P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.
Activation of 6‐phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus
Authors: Nariya Hirofumi , Inouye Sumiko 
Published: March 2003

Summary Myxococcus xanthus is a Gram‐negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic‐like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6‐phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx‐PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx‐PFK were expressed in Escherichia coli and purified. Mx‐PFK was found to be phosphorylated by Pkn4 at Thr‐226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx‐PFK enhanced its activity 2.7‐fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx‐PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx‐PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.
Proteome analysis of Escherichia coli K‐12 by two‐dimensional native‐state chromatography and MALDI‐MS
Authors: Champion Matthew M. , Campbell Christopher S. , Siegele Deborah A. , Russell David H. , Hu James C. 
Published: March 2003

SummaryTo identify proteins expressed in Escherichia coli K‐12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native‐state high‐performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first‐dimension fractions to proteins visualized by two‐dimensional gel electrophoresis. Co‐fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high‐throughput gel‐independent identification of proteins. It can also be used to assign identities to spots visualized by two‐dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.
Differential analysis of DNA microarray gene expression data
Authors: Hatfield G. Wesley , Hung She‐pin , Baldi Pierre 
Published: March 2003

SummaryHere, we review briefly the sources of experimental and biological variance that affect the interpretation of high‐dimensional DNA microarray experiments. We discuss methods using a regularized t‐test based on a Bayesian statistical framework that allow the identification of differentially regulated genes with a higher level of confidence than a simple t‐test when only a few experimental replicates are available. We also describe a computational method for calculating the global false‐positive and false‐negative levels inherent in a DNA microarray data set. This method provides a probability of differential expression for each gene based on experiment‐wide false‐positive and ‐negative levels driven by experimental error and biological variance.
Penicillin‐binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid‐cell in comparison with the old cell pole
Authors: Den Blaauwen Tanneke , Aarsman Mirjam E. G. , Vischer Norbert O. E. , Nanninga Nanne 
Published: March 2003

SummaryThe localization of penicillin‐binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)–PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot‐like pattern and also at mid‐cell during cell division. PBP2 disappeared from mid‐cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid‐cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid‐cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.
A new Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic β‐oxidation pathway
Authors: Campbell John W. , Morgan‐Kiss Rachael M. , E. Cronan John 
Published: March 2003

Summary Escherichia coli
uses fatty acids as a sole carbon and energy source during aerobic growth by means of the enzymes encoded by the
fad
regulon. We report that this bacterium can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor such as nitrate is available. This anaerobic utilization pathway is distinct from the well‐studied aerobic pathway in that (i) it proceeds normally in mutant strains lacking various enzymes of the aerobic pathway; (ii) it functions with fatty acids (octanoate and decanoate) that cannot be used by wild‐type
E. coli
strains under aerobic conditions; and (iii) super‐repressor mutants of the
fadR
regulatory locus that block aerobic growth on fatty acids fail to block the anaerobic pathway. We have identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty acids. A strong FadR binding site was detected upstream of the
yfcY
gene consistent with microarray analyses, indicating that
yfcYX
expression is negatively regulated by FadR under aerobic growth conditions. In contrast, transcriptional regulation of
ydiD
appears to be independent of FadR, and anaerobic growth on fatty acids is not under FadR control. These three genes are conserved in the available genome sequences of pathogenic
E. coli
,
Shigella
and
Salmonella strains.
An extended role for the nucleoid structuring protein H‐NS in the virulence gene regulatory cascade of Shigella flexneri
Authors: Beloin Christophe , Dorman Charles J. 
Published: March 2003

SummaryThe H‐NS nucleoid structuring protein has been shown previously to play a negative role in controlling virulence gene expression in Shigella flexneri by repressing transcription of the virF and virB regulatory genes and the VirF‐dependent icsA structural gene under non‐permissive growth conditions. Here, we show that H‐NS also acts at the promoters of the VirB‐dependent structural genes in the regulatory cascade. H‐NS protein binds to the promoter regions in vivo and in vitro. The promoters were shown physically and by in silico analysis to contain regions of DNA curvature, a feature of H‐NS binding sites. H‐NS binding sites were determined by DNase I footprinting at the icsB and the virA promoters. The locations of these sites were consistent with a role for H‐NS as a transcription repressor. The VirB‐dependent structural gene promoters were found to respond directly to the H‐NS repressor, revealing a level of control that is additional to that exerted by the H‐NS‐dependent virB activator gene. Moreover, the promoters were sensitive to the level of VirB protein in the cell, requiring a threshold level of VirB to be reached before becoming active. A model is discussed in which the levels of expression of the structural genes reflect the outcome of competition between the countervailing regulatory activities of the H‐NS and VirB proteins.
Linear plasmid SLP2 of Streptomyces lividans is a composite replicon
Authors: Huang Chih‐Hung , Chen Chung‐Yung , Tsai Hsiu‐Hui , Chen Chi , Lin Yi‐Shing , Chen Carton W. 
Published: March 2003

SummarySLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The  rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase‐like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini‐linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.
Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA
Authors: Milohanic Eliane , Glaser Philippe , Coppée Jean‐Yves , Frangeul Lionel , Vega Yolanda , Vázquez‐Boland José A. , Kunst Frank , Cossart Pascale , Buchrieser Carmen 
Published: March 2003

SummaryPrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole‐genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild‐type EGDe and a prfA‐deleted mutant (EGDe ΔprfA). Both strains were grown at 37°C in brain–heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178–lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA‐deleted mutant (P14ΔprfA) and the EGDe strain revealed interesting strain‐specific differences. Sequences strongly similar to a sigma B‐dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A‐dependent promoter. In contrast, a second set of PrfA‐regulated genes lack a PrfA box and are expressed from a sigma B‐dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.