bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the Rho GTPases in neuronal development
Authors: Govek Eve-Ellen , Newey Sarah E. , Van Aelst Linda
Published: January 2005

Our brain serves as a center for cognitive function and neurons within the brain relay and store information about our surroundings and experiences. Modulation of this complex neuronal circuitry allows us to process that information and respond appropriately. Proper development of neurons is therefore vital to the mental health of an individual, and perturbations in their signaling or morphology are likely to result in cognitive impairment. The development of a neuron requires a series of steps that begins with migration from its birth place and initiation of process outgrowth, and ultimately leads to differentiation and the formation of connections that allow it to communicate with appropriate targets. Over the past several years, it has become clear that the Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance. Given the importance of these molecules in these processes, it is therefore not surprising that mutations in genes encoding a number of regulators and effectors of the Rho GTPases have been associated with human neurological diseases. This review will focus on the role of the Rho GTPases and their associated signaling molecules throughout neuronal development and discuss how perturbations in Rho GTPase signaling may lead to cognitive disorders.
The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin
Authors: Swaminathan Jyothishmathi , Baxter Ellen M. , Corces Victor G.
Published: January 2005

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. Here we show that the histone H2A variant of Drosophila melanogaster, H2Av, localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. H2Av accumulation or histone H4 Lys 12 acetylation is not affected by mutations in Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin and requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place.
Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms
Authors: Kahvejian Avak , Svitkin Yuri V. , Sukarieh Rami , M'Boutchou Marie-Noël , Sonenberg Nahum
Published: January 2005

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (∼65% vs. ∼35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5′-end of mRNA.
Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export
Authors: Dunn Ewan F. , Hammell Christopher M. , Hodge Christine A. , Cole Charles N.
Published: January 2005

In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export. Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription. A poly(A) tail added by the normal 3′-processing machinery is necessary but not sufficient for export. Retention depends on the exosome. In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3′ processing to export. Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm. It can exit the nucleus either as cargo of exportin 1 or bound to mRNA. Pab1 is essential but several bypass suppressors have been identified. Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription. Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs. The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.
The FLP proteins act as regulators of chlorophyll synthesis in response to light and plastid signals in Chlamydomonas
Authors: Falciatore Angela , Merendino Livia , Barneche Fredy , Ceol Mauro , Meskauskiene Rasa , Apel Klaus , Rochaix Jean-David
Published: January 2005

In photosynthetic organisms the accumulation of harmful photodynamic chlorophyll precursors is prevented because of the tight regulation of the tetrapyrrole pathway. FLU is one of the regulatory factors involved in this process in land plants. We have examined the function of a Flu-like gene (FLP) from Chlamydomonas that gives rise to two FLP transcripts through alternative splicing. These transcripts are translated into a short and a long protein that differ by only 12 amino acids but that interact differently with glutamyl-tRNA reductase, an enzyme involved in an early step of the chlorophyll biosynthetic pathway. Expression of FLPs is light-regulated at the level of RNA accumulation and splicing and is altered by mutations affecting the pathway. The relative levels of the long and short forms of FLP can be correlated with the accumulation of specific porphyrin intermediates, some of which have been implicated in a signaling chain from the chloroplast to the nucleus. Reciprocally, reduction of the FLP proteins by RNA interference leads to the accumulation of several porphyrin intermediates and to photobleaching when cells are transferred from the dark to the light. Thus the FLP proteins act as regulators of chlorophyll synthesis, and their expression is controlled by light and plastid signals.
Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis
Authors: Gilchrist C. A. , Gray D. A. , Stieber A. , Gonatas N. K. , Kopito R. R.
Published: January 2005

The ubiquitin‐proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G93A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild‐type or dominant‐negative mutant ubiquitin (UbK48R). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9–12% of total ubiquitin. Mice expressing the G93A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G93A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild‐type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.
Comprehensive survey of carapacial ridge‐specific genes in turtle implies co‐option of some regulatory genes in carapace evolution
Authors: Kuraku Shigehiro , Usuda Ryo , Kuratani Shigeru
Published: January 2005

Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft‐shelled turtle, Pelodiscus sinensis, using microbead‐based differential cDNA analysis and real‐time reverse transcription‐polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid‐binding protein‐I (CRABP‐I), adenomatous polyposis coli down‐regulated 1 (APCDD1), and lymphoid enhancer‐binding factor‐1 (LEF‐1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF‐1, the nuclear localization of β‐catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co‐option of pre‐existing genes.
Gene expression and digit homology in the chicken embryo wing
Authors: Welten Monique C. M. , Verbeek Fons J. , Meijer Annemarie H. , Richardson Michael K.
Published: January 2005

SummaryThe bird wing is of special interest to students of homology and avian evolution. Fossil and developmental data give conflicting indications of digit homology if a pentadactyl “archetype” is assumed. Morphological signs of a vestigial digit I are seen in bird embryos, but no digit‐like structure develops in wild‐type embryos. To examine the developmental mechanisms of digit loss, we studied the expression of the high‐mobility group box containingSox9gene, and bone morphogenetic protein receptor 1b (bmpR‐1b)—markers for precondensation and prechondrogenic cells, respectively. We find an elongated domain ofSox9expression, but nobmpR‐1bexpression, anterior to digit II. We interpret this as a digit I domain that reaches precondensation, but not condensation or precartilage stages. It develops late, when the tissue in which it is lodged is being remodeled. We consider these findings in the light of previousHoxd‐11misexpression studies. Together, they suggest that there is a digit I vestige in the wing that can be rescued and undergo development if posterior patterning cues are enhanced. We observedSox9expression in the elusive “element X” that is sometimes stated to represent a sixth digit. Indeed, incongruity between digit domains and identities in theropods disappears if birds and other archosaurs are considered primitively polydactyl. Our study provides the first gene expression evidence for at least five digital domains in the chick wing. The failure of the first to develop may be plausibly linked to attenuation of posterior signals.
Development and phenotypic correlations: the evolution of tooth shape in Sorex araneus
Authors: Polly P. David
Published: January 2005

Summary
Do morphogenetic processes cause common patterns of phenotypic covariation, and do those patterns evolve over microevolutionary timescales? Evolution of molar shape variance–covariance (P) matrixes was studied in five populations of the common shrew, Sorex araneus. P matrix evolution was assessed using matrix correlation, matrix disparity, and common principal component analysis (CPCA). Significant changes in covariance structure were found among the populations, but the differences were small. A computer model was used to estimate the theoretical covariance introduced into the phenotype by developmental interactions. Molar developmental processes explained some of the covariance in the shrew samples, especially as measured by matrix correlation, but the proportion was relatively small. Developmental principal components (PCs) were only infrequently associable with common principal components. The results suggest that molar shape P matrixes can evolve quickly in a manner only loosely constrained by development, and that their shared covariance is probably dominated by factors more proximate than development. Rarefaction showed that sample size severely affected P comparisons when n < 15 for matrix correlation and disparity, and when n < 30 for CPCA. Among CPCA evaluation criteria, Akaike Information Criterion performed better than jump‐up at n < 30, but worse at n > 30.
Abd‐B expression in Porcellio scaber Latreille, 1804 (Isopoda: Crustacea): conserved pattern versus novel roles in development and evolution
Authors: Brena C. , Liu P. Z. , Minelli A. , Kaufman T. C.
Published: January 2005

Summary
The Hox genes are intimately involved in patterning the animal body during development and are considered to have had a pivotal role in the evolution of different body plans among the metazoans. From this perspective, crustaceans, a group that has evolved an extreme diversity of body structures, represent a choice group in which to study the evolution of these genes and their expression. The expression of one of these genes, Abdominal‐B (Abd‐B), has only been studied in two distantly related crustaceans, Artemia and Sacculina, where it shows dissimilar patterns, highly differentiated from the one described in other arthropods. Moreover, we have no information for the Malacostraca. Thus, we cloned the gene Abd‐B and followed its expression through development by in situ hybridization in the isopod Porcellio scaber. We found a highly dynamic expression pattern of PsAbd‐B during embryonic development. In early stages, it is expressed in the posterior‐most part of the germ band, in a domain common to several arthropods studied to date, and later it is expressed in the developing limb buds of the pleon and still later in the endopodites of the third to fifth pleopodites. This raises the interesting possibility of the involvement of this gene in the later respiratory specialization of these appendages. In association with the above expression domain, Abd‐B appears to be expressed in later stages also in the ventral ectoderm, raising the further suggestion of its possible involvement in patterning the developing nervous system. Moreover, we show that the first pleopod and the endopodite of the second pleopod, whereas present as limb buds in early embryonic stages, are later reduced and actually absent in the first postembryonic stage, although they reappear again in adults. These appendages thus represent an example of Lazarus appendages. Our data show strong plasticity in the use of a key developmental gene and point out the necessity of further research that may end with a revision of the current understanding of its role in animal evolution.
Evolution of dorsal–ventral axis formation in arthropod appendages: H15 and optomotor‐blind/bifid‐type T‐box genes in the millipede Glomeris marginata (Myriapoda: Diplopoda)
Authors: Prpic Nikola‐Michael , Janssen Ralf , Damen Wim G. M. , Tautz Diethard
Published: January 2005

Summary
In Drosophila, the T‐box genes optomotor‐blind (omb) and H15 have been implicated in specifying the development of the dorso–ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. Here we extend the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe‐shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene we were able to identify two paralogous genes in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm‐H15‐1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear.
Role of the male BmDSX protein in the sexual differentiation of Bombyx mori
Authors: Suzuki Masataka G. , Funaguma Shunsuke , Kanda Toshio , Tamura Toshiki , Shimada Toru
Published: January 2005

Summary
The sex determination pathway is different between Drosophila melanogaster and Bombyx mori in the initial signal. Here we show evidence that the sex determination pathway in B. mori is similar to that of D. melanogaster at the level of the terminal regulator, doublesex (dsx), which is essential for the proper differentiation of the sexually dimorphic somatic features of D. melanogaster. In B. mori, a homolog of dsx (Bmdsx) is expressed in various tissues, and its primary transcript is alternatively spliced in males and females to yield sex‐specific mRNAs that encode male‐specific (BmDSXM) and female‐specific (BmDSXF) polypeptides. In the studies reported here, transgenic silkworms carrying a construct with a Bmdsx male cDNA placed under the control of either an hsp70 promoter or a Bombyx actin3 promoter were generated by piggyBac‐mediated germline transformation. Ectopic expression of the male cDNA in females resulted in abnormal differentiation of certain female‐specific genital organs and caused partial male differentiation in female genitalia. Transgenic analysis also revealed that the expression of BmDSXM in females caused repression of the female‐specifically expressed gene, the vitellogenin gene, and also resulted in activation of the pheromone‐binding protein gene that is dominantly expressed in males. These results provide evidence that the role of BmDSXM includes the activation of some aspects of male differentiation as well as the repression of female differentiation. Taken together with our previous data on the function of BmDSXF, we can conclude that Bmdsx is a double‐switch gene at the final step in the sex‐determination cascade of B. mori.
Evolutionarily Divergent Extradiol Dioxygenases Possess Higher Specificities for Polychlorinated Biphenyl Metabolites
Authors: Fortin Pascal D. , Lo Andy T.-F. , Haro María-Amparo , Kaschabek Stefan R. , Reineke Walter , Eltis Lindsay D.
Published: January 2005

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from
Burkholderia
sp. strain LB400 (DHBD
LB400
), DHBD
P6
-I and DHBD
P6
-III from
Rhodococcus globerulus
P6, and 2,2′,3-trihydroxybiphenyl dioxygenase from
Sphingomonas
sp. strain RW1 (THBD
RW1
). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the
K
m
app
values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3′,5′-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBD
P6
-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBD
P6
-III than was DHB. Indeed, DHBD
P6
-III had the highest apparent specificity for 4,3′,5′-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBD
RW1
had the highest specificity for 2′-Cl DHB and was at least five times more resistant to inactivation by 2′-Cl DHB, consistent with the similarity between the latter and 2,2′,3-trihydroxybiphenyl. Nonetheless, THBD
RW1
had the lowest specificity for 2′,6′-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (
k
cat
app
< 0.001 s
−1
).
Analysis of a DtxR-Regulated Iron Transport and Siderophore Biosynthesis Gene Cluster in
Corynebacterium diphtheriae
Authors: Kunkle Carey A. , Schmitt Michael P.
Published: January 2005

This report describes a genetic locus associated with siderophore biosynthesis and transport in
Corynebacterium diphtheriae
. A BLAST search of the
C. diphtheriae
genome identified a seven-gene cluster that included four genes, designated
ciuA, ciuB, ciuC
, and
ciuD
, whose predicted products are related to ABC-type iron transporters. Downstream from
ciuD
is the
ciuE
gene, whose predicted product is similar to the aerobactin biosynthetic enzymes IucA and IucC. The CiuE protein, which has a predicted mass of 121,582 Da and is approximately twice the size of either IucC or IucA, is homologous to each of these proteins in both its N- and C-terminal regions.
C. diphtheriae ciuE
deletion mutants exhibited a defect in siderophore production, iron uptake, and growth in low-iron medium. Mutations in the
ciuA
gene, whose predicted product is a lipoprotein component of an iron transport system, resulted in a severe defect in iron uptake and reduced ability to use the
C. diphtheriae
siderophore as an iron source. Site-directed mutations in
irp6A
, a gene previously reported to be associated with siderophore transport, had no effect on iron uptake or the utilization of the
C. diphtheriae
siderophore as an iron source. Transcriptional analysis demonstrated that expression of
ciuA
and
ciuE
is DtxR and iron regulated, and DNase I protection experiments confirmed the presence of DtxR binding sites upstream from each of these genes. Thus, this iron- and DtxR-regulated gene cluster is involved in the synthesis and transport of the
C. diphtheriae
siderophore.
Identification ofChlamydia trachomatisGenomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA)
Authors: Rau Annette , Wyllie Susan , Whittimore Judy , Raulston Jane E.
Published: January 2005

TheChlamydia trachomatisdivalent cation-dependent regulator (DcrA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators. Chlamydial DcrA specifically binds to a consensusEscherichia coliFur box and is able to complement anE. coliFur mutant. In this report, theE. coliFur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized byE. coliFur. The predictive regulatory regions of 28C. trachomatisopen reading frames contained sequences functionally recognized byE. coliFur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways. Selected FURTA-positive sequences were subsequently examined for recognition byC. trachomatisDcrA using an electrophoretic mobility shift assay. The resultant data show thatC. trachomatisDcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DcrA and the Fur family of regulators.
Error-Prone DNA Polymerase IV Is Regulated by the Heat Shock Chaperone GroE in
Escherichia coli
Authors: Layton Jill C. , Foster Patricia L.
Published: January 2005

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The
groE
operon consists of two genes,
groES
and
groEL
; point mutations in either gene conferred the same phenotype, reducing Lac
+
adaptive mutation 10- to 20-fold.
groE
mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In
recG
+
strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in
recG
mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.
The
LEE1
Promoters from both Enteropathogenic and Enterohemorrhagic
Escherichia coli
Can Be Activated by PerC-Like Proteins from Either Organism
Authors: Porter Megan E. , Mitchell Paul , Free Andrew , Smith David G. E. , Gally David L.
Published: January 2005

The PerC protein of enteropathogenic
Escherichia coli
(EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the
LEE1
promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic
E. coli
(EHEC) lacks PerC-dependent activation due to utilization of an alternative
LEE1
promoter and lack of a
perC
gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC
LEE1
promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The
perC1
genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual
perC1
copies appear to be poorly expressed, the total
perC1
expression level from a strain encoding multiple copies approaches that of
perC
in EPEC and may therefore contribute significantly to
LEE1
activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the
LEE1
promoter, suggesting that other core
E. coli
proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.
Mutation Analysis of the Histidine Residues in the Glycylglycine Endopeptidase ALE-1
Authors: Fujiwara Tamaki , Aoki Shin , Komatsuzawa Hitoshi , Nishida Tetsuya , Ohara Masaru , Suginaka Hidekazu , Sugai Motoyuki
Published: January 2005

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by
Staphylococcus capitis
EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (ΔN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of ΔN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of ΔN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant ΔN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.
Posttranslational Regulation of Nitrate Assimilation in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Kobayashi Masaki , Takatani Nobuyuki , Tanigawa Mari , Omata Tatsuo
Published: January 2005

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium
Synechocystis
sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the
nrtABCD
genes was completely inhibited by ammonium as in
Synechococcus elongatus
strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the
Synechococcus
strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the P
II
protein (
glnB
gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the
Synechococcus
strain. Mutants expressing the P
II
derivatives in which Ala or Glu is substituted for the conserved Ser
49
, which has been shown to be the phosphorylation site in the
Synechococcus
strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in
Synechococcus
either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the P
II
protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of P
II
, as was previously reported for
Synechococcus elongatus
strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.
Probing Direct Interactions between CodY and theoppDPromoter ofLactococcus lactis
Authors: den Hengst Chris D. , Curley Peter , Larsen Rasmus , Buist Girbe , Nauta Arjen , van Sinderen Douwe , Kuipers Oscar P. , Kok Jan
Published: January 2005

CodY ofLactococcus lactisMG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes includepepN,pepC,opp-pepO1, and probablyprtPM,pepX, andpepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate thatL. lactisCodY interacts directly with a region upstream of the promoter of its major target known so far, theoppsystem. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region ofoppDyielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.
Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation
Authors: Adams Thorsten M. , Wentzel Alexander , Kolmar Harald
Published: January 2005

Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, β-lactamase inhibitor protein, or β-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.
Analysis of the Precursor rRNA Fractions of Rapidly Growing Mycobacteria: Quantification by Methods That Include the Use of a Promoter (
rrnA
P1) as a Novel Standard
Authors: Menéndez María del Carmen , Rebollo María José , Núñez María del Carmen , Cox Robert A. , García María Jesús
Published: January 2005

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their
rrn
operons.
Mycobacterium chelonae
and
M. fortuitum
are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two
rrn
operons per genome. Quantification of precursor
rrn
transcriptional products (pre-
rrn)
has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first
rrnA
promoter in both species, namely,
rrnA
P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when
M. fortuitum
infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its
rrn
operons is preferentially used under such conditions.
Caulobacter crescentusRequires RodA and MreB for Stalk Synthesis and Prevention of Ectopic Pole Formation
Authors: Wagner Jennifer K. , Galvani Cheryl D. , Brun Yves V.
Published: January 2005

Caulobacter crescentuscells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 inEscherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, inC. crescentus, therodAgene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity inC. crescentus.
Staphylococcus aureus
Serves as an Iron Source for
Pseudomonas aeruginosa
during In Vivo Coculture
Authors: Mashburn Lauren M. , Jett Amy M. , Akins Darrin R. , Whiteley Marvin
Published: January 2005

Pseudomonas aeruginosa
is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with
P. aeruginosa
alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of
P. aeruginosa
in vivo, and less is known about the impact of coinfecting bacteria on
P. aeruginosa
pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of
P. aeruginosa
in monoculture and in coculture with
Staphylococcus aureus
. Monoculture results indicate that approximately 5% of all
P. aeruginosa
genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of
S. aureus
caused decreased transcription of
P. aeruginosa
iron-regulated genes during in vivo coculture, indicating that the presence of
S. aureus
increases usable iron for
P. aeruginosa
in this environment. We propose a model where
P. aeruginosa
lyses
S. aureus
and uses released iron for growth in low-iron environments.
Introns in the Cytolethal Distending Toxin Gene ofActinobacillus actinomycetemcomitans
Authors: Tan Kai Soo , Ong Grace , Song Keang Peng
Published: January 2005

In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria.Actinobacillus actinomycetemcomitansis a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within thecdtgene ofA. actinomycetemcomitans. By use of reverse transcription-PCR,cdttranscripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carryingcdtwas cloned intoEscherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of thecdtRNA was located within thecdtCgene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in bothcisandtransconfigurations.
Molecular Nature of Spontaneous Modifications ingacSWhich Cause Colony Phase Variation inPseudomonassp. Strain PCL1171
Authors: van den Broek Daan , Chin-A-Woeng Thomas F. C. , Bloemberg Guido V. , Lugtenberg Ben J. J.
Published: January 2005

Pseudomonassp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations ingacAor/andgacS. Mutation ofgacreduced both the length of the lag phase and the generation time. Isolation and sequencing of thegacSgenes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-typegacAandgacSgenes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in eithergacAorgacS. The reversion ofgacAorgacSto the wild type was not affected by mutation ofrecAandrecB. We conclude that inPseudomonassp. strain PCL1171, mutations ingacAandgacSare the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations ingacresult in dynamic switches between the “wild-type” population and the subpopulations harboring spontaneous mutations ingacAand orgacS, thereby enabling both populations to be maintained.
Roles of Thioredoxin Reductase during the Aerobic Life of
Lactococcus
lactis
Authors: Vido Karin , Diemer Hélène , Van Dorsselaer Alain , Leize Emmanuelle , Juillard Vincent , Gruss Alexandra , Gaudu Philippe
Published: January 2005

Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including
Lactococcus
lactis
, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an
L. lactis
trxB1
mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the
trxB1
mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the
trxB1
mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the
trxB1
mutant. Two additional effects of
trxB1
were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in
L. lactis
differed by the oxidation state of catalytic-site cysteine C
152
. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the
trxB1
mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in
L. lactis
and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for
trxB1
deficiency is suggested.
Analysis of MinD Mutations Reveals Residues Required for MinE Stimulation of the MinD ATPase and Residues Required for MinC Interaction
Authors: Zhou Huaijin , Schulze Ryan , Cox Sandra , Saez Cristian , Hu Zonglin , Lutkenhaus Joe
Published: January 2005

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.
The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer
Authors: Matson Steven W. , Ragonese Heather
Published: January 2005

The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site- and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5′-to-3′ helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first ∼310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraIΔ252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraIΔ252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraIΔ252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.