bluuebunny/crossref_metadata_2025_split huggingface dataset:
Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase
Authors: Stickney Leigh M. , Hankins Janet S. , Miao Xin , Mackie George A. 
Published: October 2005

ABSTRACT
We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.
Structure and Increased Thermostability ofRhodococcussp. Naphthalene 1,2-Dioxygenase
Authors: Gakhar Lokesh , Malik Zulfiqar A. , Allen Christopher C. R. , Lipscomb David A. , Larkin Michael J. , Ramaswamy S. 
Published: October 2005

ABSTRACTRieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase fromRhodococcussp. strain NCIMB12038. The structure of theRhodococcusenzyme reveals that, despite a ∼30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 Å. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to theRhodococcusenzyme in the same orientation as in thePseudomonasenzyme. Circular dichroism spectroscopy experiments show that theRhodococcusenzyme has higher thermostability than the naphthalene dioxygenase fromPseudomonasspecies. ThePseudomonasenzyme has an apparent melting temperature of 55°C while theRhodococcusenzyme does not completely unfold even at 95°C. Both enzymes, however, show similar unfolding behavior in urea, and theRhodococcusenzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of theRhodococcusenzyme may be attributed to a larger buried surface area and extra salt bridge networks between the α and β subunits in theRhodococcusenzyme.
Transcriptome and Physiological Responses to Hydrogen Peroxide of the Facultatively Phototrophic Bacterium
Rhodobacter sphaeroides
Authors: Zeller Tanja , Moskvin Oleg V. , Li Kuanyu , Klug Gabriele , Gomelsky Mark 
Published: October 2005

ABSTRACT

The transcriptome responses to hydrogen peroxide, H
2
O
2
, of the facultatively phototrophic bacterium
Rhodobacter sphaeroides
grown under semiaerobic conditions were investigated. At 7 min after the addition of 1 mM H
2
O
2
, the expression of approximately 9% of all genes (total, 394) was changed reliably by at least twofold. At 30 min, the number of genes (total, 88) and the magnitude of expression changes were much lower, indicating rapid recovery from stress. Two types of responses were observed: (i) an H
2
O
2
stress response per se and (ii) a shift to high-oxygen metabolism. The former response involved the upregulation of genes for H
2
O
2
detoxification, protein folding and proteolysis, DNA damage repair, iron transport and storage, iron-sulfur cluster repair, and the downregulation of genes for protein translation, motility, and cell wall and lipopolysaccharide synthesis. The shift to high-oxygen metabolism was evident from the differential regulation of genes for aerobic electron transport chain components and the downregulation of tetrapyrrole biosynthesis and photosystem genes. The abundance of photosynthetic complexes was decreased upon prolonged exposure of
R. sphaeroides
to H
2
O
2
, thus confirming the physiological significance of the transcriptome data. The regulatory pathways mediating the shift to high-oxygen metabolism were investigated. They involved the anaerobic activator FnrL and the antirepressor-repressor AppA-PpsR system. The transcription of FnrL-dependent genes was down at 7 min, apparently due to the transient inactivation by H
2
O
2
of the iron-sulfur cluster of FnrL. The transcription of the AppA-PpsR-dependent genes was down at 30 min, apparently due to the significant decrease in
appA
mRNA.
Functional Consequences of Genome Evolution inListeria monocytogenes: the lmo0423 and lmo0422 Genes Encode σCand LstR, a Lineage II-Specific Heat Shock System
Authors: Zhang Chaomei , Nietfeldt Joe , Zhang Min , Benson Andrew K. 
Published: October 2005

ABSTRACTListeria monocytogenesstrains belonging to phylogenetic lineage II (serotypes 1/2a, 1/2c, and 3a) carry a lineage-specific genome segment encoding a putative sigma subunit of RNA polymerase (lmo0423, herein referred to assigC), a gene of unknown function (lmo0422) similar to thepadRfamily of regulators, and a gene that is similar to therodA-ftsWfamily of cell wall morphology genes (lmo0421). To understand the function of this set of genes, their expression patterns and the effects of null mutations in the lineage IIL. monocytogenesstrain 10403S were examined. The data are consistent with the three genes comprising an operon (thesigCoperon) that is highly induced by temperature upshift. The operon is transcribed from three different promoters, the proximal of which (P1) depends uponsigCitself. Null mutations insigCor lmo0422 increase the death rate at lethal temperatures and cause loss of thermal adaptive response, whereas the lmo0421 mutation causes only a loss of the adaptive response component. Only thesigCmutation affects transcription from the P1 promoter, whereas ectopic expression of lmo0422 from the PSPACpromoter complements the individual lmo0422 andsigCnull mutations, showing that lmo0422 is the actual thermal resistance regulator or effector whilesigCprovides a mechanism for temperature-dependent transcription of lmo0422 from P1. Our genetic and phylogenetic analyses are consistent with lmo0422—renamedlstR(for lineage-specific thermal regulator)—andsigCcomprising a system of thermal resistance that was ancestral to the genusListeriaand was subsequently lost during divergence of the lineage IL. monocytogenespopulation.
A Glycosyltransferase Involved in Biosynthesis of Triglycosylated Glycopeptidolipids inMycobacterium smegmatis: Impact on Surface Properties
Authors: Deshayes Caroline , Laval Françoise , Montrozier Henri , Daffé Mamadou , Etienne Gilles , Reyrat Jean-Marc 
Published: October 2005

ABSTRACTThe cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. InMycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.
Whole-Genome Sequencing of
Staphylococcus haemolyticus
Uncovers the Extreme Plasticity of Its Genome and the Evolution of Human-Colonizing Staphylococcal Species
Authors: Takeuchi Fumihiko , Watanabe Shinya , Baba Tadashi , Yuzawa Harumi , Ito Teruyo , Morimoto Yuh , Kuroda Makoto , Cui Longzhu , Takahashi Mikio , Ankai Akiho , Baba Shin-ichi , Fukui Shigehiro , Lee Jean C. , Hiramatsu Keiichi 
Published: October 2005

ABSTRACT

Staphylococcus haemolyticus
is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of
S.haemolyticus
to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of
S.haemolyticus
,
Staphylococcus aureus
, and
Staphylococcus epidermidis
were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the “
oriC
environ,” likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of
S.haemolyticus
,
S.aureus
, and
S.epidermidis
elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the
S.haemolyticus
chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.
Defining Genes in the Genome of the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Implications for All Microbial Genomes
Authors: Poole Farris L. , Gerwe Brian A. , Hopkins Robert C. , Schut Gerrit J. , Weinberg Michael V. , Jenney Francis E. , Adams Michael W. W. 
Published: October 2005

ABSTRACT

The original genome annotation of the hyperthermophilic archaeon
Pyrococcus furiosus
contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of
P. furiosus
. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in
P. furiosus
when grown at either 95 or 72°C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in
Escherichia coli
, although only one of the seven ORFs was expressed in
P. furiosus
under the growth conditions tested. It is concluded that the
P. furiosus
genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.
Identification of the
rctA
Gene, Which Is Required for Repression of Conjugative Transfer of Rhizobial Symbiotic Megaplasmids
Authors: Pérez-Mendoza Daniel , Sepúlveda Edgardo , Pando Victoria , Muñoz Socorro , Nogales Joaquina , Olivares José , Soto Maria J. , Herrera-Cervera José A. , Romero David , Brom Susana , Sanjuán Juan 
Published: October 2005

ABSTRACT

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of
Rhizobium etli
, has revealed a novel gene,
rctA
, as an essential element of a regulatory system for silencing the conjugative transfer of
R. etli
pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The
rctA
gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors,
rctA
transcription seems to be positively autoregulated.
rctA
expression is greatly reduced upon overexpression of another gene,
rctB
, previously identified as a putative activator of
R. etli
pSym conjugal transfer. Thus,
rctB
seems to counteract the repressive action of
rctA. rctA
homologs are present in at least three other bacterial genomes within the order
Rhizobiales
, where they are invariably located adjacent to and divergently transcribed from putative
virB
-like operons. We show that similar to that of
R. etli
pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of
Sinorhizobium meliloti
is also subjected to the inhibitory action of
rctA
. Our data provide strong evidence that the
R. etli
and
S. meliloti
pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the
rctA
function. The
rctA
gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order
Rhizobiales
.
Type III Restriction Is Alleviated by Bacteriophage (RecE) Homologous Recombination Function but Enhanced by Bacterial (RecBCD) Function
Authors: Handa Naofumi , Kobayashi Ichizo 
Published: October 2005

ABSTRACT

Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an
Escherichia coli
host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage—presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions
recE
and
recT
of Rac prophage. This type III restriction alleviation does not depend on
lar
on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.
Ribose-5-Phosphate Biosynthesis in
Methanocaldococcus jannaschii
Occurs in the Absence of a Pentose-Phosphate Pathway
Authors: Grochowski Laura L. , Xu Huimin , White Robert H. 
Published: October 2005

ABSTRACT

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry
43:
7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in
Methanocaldococcus jannaschii
, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-
2
H
2
]- and [U-
13
C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of
d
-
arabino
-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the
d
-
ara-bino
-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from
d
-
arabino
-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.
σ
B
Activity in a
Staphylococcus aureus hemB
Mutant
Authors: Senn Maria M. , Bischoff Markus , von Eiff Christof , Berger-Bächi Brigitte 
Published: October 2005

ABSTRACT

Inactivation of
hemB
in
Staphylococcus aureus
strain Newman resulted in a small-colony phenotype and was accompanied by an altered expression pattern of global regulators and control of virulence factor production. Transcription profiles followed over 15 h by Northern blot analyses revealed that transcripts of the global regulators
arl
,
rot
,
sae
,
sarR
,
sarS
,
srr
,
svrA
, and
sigB
disappeared after the exponential phase and that both
agr
transcripts were completely absent in the
hemB
mutant. Apart from a general concentration of transcriptional activity to the exponential phase, premature gene expression was observed for
rot
,
hla
, and
spa
. Nevertheless, reported σ
B
-dependent transcripts, such as
sarC
and
clfA
, were produced throughout the 15-h growth period monitored. The absence of these transcripts in a
hemB sigB
double mutant demonstrated their dependence on σ
B
and indicated an unexpected, permanent σ
B
activity in the
hemB
mutant. Variations in the extents of the directly σ
B
-controlled
asp23
,
rsbVW-sigB
, and
sarC
transcripts argue for additional factors modulating σ
B
activity. This study provides the first extended synopsis of the transcriptional patterns of different regulators over the entire growth cycle in the widely used Newman strain.
Resolvase-In Vivo Expression Technology Analysis of theSalmonella entericaSerovar Typhimurium PhoP and PmrA Regulons in BALB/c Mice
Authors: Merighi Massimo , Ellermeier Craig D. , Slauch James M. , Gunn John S. 
Published: October 2005

ABSTRACTSalmonella entericamodulates resistance to antimicrobial peptides in part via covalent modifications of the lipopolysaccharide (LPS). The two-component systems PhoP/PhoQ and PmrA/PmrB are activated during infection and regulate several genes involved in LPS modifications by responding to signals such as pH, iron, magnesium, and antimicrobial peptides. A recombination-based in vivo expression technology approach was adopted to analyze the spatial-temporal patterns of in vivo expression of genes of the PhoP and PmrA regulons and to identify the in vivo signals modulating their transcription. In vitro, we showed PhoP- and/or PmrA-dependent induction ofpmrH(LPS aminoarabinose modification operon) by acidic pH, low levels of magnesium, or high levels of Fe(III). Upregulation in cultured J774A.1 macrophages was shown forpmrH,pagP(LPS palmitate addition), andssaB(pathogenicity island II secretion) but not forprgH(pathogenicity island I secretion). Increased levels ofpmrH,phoP, andprgHtranscription but notssaBwere observed in bacteria isolated from the lumen of the distal ileum. Bacteria isolated from spleens of orally inoculated mice showed no further induction ofprgHbut had the highest expression ofpmrH,pagP, andssaB. In vivo induction ofpmrHwas fully dependent onpmrAandphoP, and buffering stomach acidity, iron chelation, or low-iron diets did not affect the expression ofpmrHin the intestinal lumen. The observation ofpmrHandpagPexpression in the intestine refutes the paradigm of PhoP/PhoQ and PmrA/PmrB in vivo expression as solely intracellularly induced and supports previous data demonstrating peroral virulence attenuation ofpmrHmutants.
NspS, a Predicted Polyamine Sensor, Mediates Activation of
Vibrio cholerae
Biofilm Formation by Norspermidine
Authors: Karatan Ece , Duncan Tammi R. , Watnick Paula I. 
Published: October 2005

ABSTRACT

Vibrio cholerae
is both an environmental bacterium and a human intestinal pathogen. The attachment of bacteria to surfaces in biofilms is thought to be an important feature of the survival of this bacterium both in the environment and within the human host. Biofilm formation occurs when cell-surface and cell-cell contacts are formed to make a three-dimensional structure characterized by pillars of bacteria interspersed with water channels. In monosaccharide-rich conditions, the formation of the
V. cholerae
biofilm requires synthesis of the VPS exopolysaccharide. MbaA (locus VC0703), an integral membrane protein containing a periplasmic domain as well as cytoplasmic GGDEF and EAL domains, has been previously identified as a repressor of
V. cholerae
biofilm formation. In this work, we have studied the role of the protein NspS (locus VC0704) in
V. cholerae
biofilm development. This protein is homologous to PotD, a periplasmic spermidine-binding protein of
Escherichia coli
. We show that the deletion of
nspS
decreases biofilm development and transcription of exopolysaccharide synthesis genes. Furthermore, we demonstrate that the polyamine norspermidine activates
V. cholerae
biofilm formation in an MbaA- and NspS-dependent manner. Based on these results, we propose that the interaction of the norspermidine-NspS complex with the periplasmic portion of MbaA diminishes the ability of MbaA to inhibit
V. cholerae
biofilm formation. Norspermidine has been detected in bacteria, archaea, plants, and bivalves. We suggest that norspermidine serves as an intercellular signaling molecule that mediates the attachment of
V. cholerae
to the biotic surfaces presented by one or more of these organisms.