bluuebunny/crossref_metadata_2025_split huggingface dataset:
Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple
rrn
Operons
Authors: Acinas Silvia G. , Marcelino Luisa A. , Klepac-Ceraj Vanja , Polz Martin F. 
Published: April 2004

ABSTRACT

The level of sequence heterogeneity among
rrn
operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related
rrn
operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of
rrn
copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ∼40% of the organisms analyzed have either one or two operons. Among the
Archaea
, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For
Archaea
, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single
rrn
operon may lower this correction factor to ∼2.5. Divergence among operons appears to be small overall for both
Bacteria
and
Archaea
, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and
Thermoanaerobacter tengcongensis
exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.
Formation of SXT Tandem Arrays and SXT-R391 Hybrids
Authors: Burrus Vincent , Waldor Matthew K. 
Published: April 2004

ABSTRACT

SXT is an integrative and conjugative element (ICE) isolated from
Vibrio cholerae
. This ∼100-kb ICE encodes resistance to multiple antibiotics and integrates site specifically into the chromosome. SXT excises from the chromosome to form a circular but nonreplicative extrachromosomal molecule that is required for its transfer. Here we found that a significant fraction of freshly isolated SXT exconjugants contained tandem SXT arrays. There was heterogeneity in the size of the SXT arrays detected in single exconjugant colonies. Some arrays consisted of more than five SXTs arranged in tandem. These extended arrays were unstable and did not persist during serial passages. The mechanism accounting for the generation of SXT arrays is unknown; however, array formation was not dependent upon
recA
and appeared to depend on conjugative transfer. While such arrays did not alter the transfer frequency of wild-type SXT, they partially complemented the transfer deficiency of a Δ
xis
SXT mutant, which is ordinarily unable to generate the extrachromosomal intermediate required for SXT transfer. Exconjugants derived from donor strains that harbored tandem arrays of SXT and R391, an SXT-related element, contained functional hybrid elements that arose from
recA
-independent recombination between the two ICEs. Thus, arrays of SXT-related elements promote the creation of novel ICEs.
The Membrane Fluidity Sensor DesK of
Bacillus subtilis
Controls the Signal Decay of Its Cognate Response Regulator
Authors: Albanesi Daniela , Mansilla María Cecilia , de Mendoza Diego 
Published: April 2004

ABSTRACT

The
Bacillus subtilis
DesK/DesR two-component system regulates the expression of the
des
gene coding for the Δ5 acyl lipid desaturase. It is believed that a decrease in membrane lipid fluidity activates the DesK/DesR signal transduction cascade, which results in synthesis of the Δ5 acyl lipid desaturase and desaturation of membrane phospholipids. These newly synthesized unsaturated fatty acids then act as negative signals of
des
transcription, thus generating a regulatory metabolic loop that optimizes membrane fluidity. We previously suggested that DesK is a bifunctional enzyme with both kinase and phosphatase activities that could assume different signaling states in response to changes in the fluidity of membrane lipids. However, no direct experimental evidence supported this proposed model. In this study, we show that the C-terminal fragment of the DesK protein (DesKC) indeed acts as an autokinase. Addition of the response regulator DesR to phosphorylated DesKC resulted in rapid transfer of the phosphoryl group to DesR. Further, phosphorylated DesR can be dephosphorylated in the presence of DesKC, thus demonstrating that the sensor kinase has the ability to covalently modify DesR through both kinase and phosphatase activities. We also present evidence that DesKC might be locked in a kinase-dominant state in vivo and that its activities are not affected either in vivo or in vitro by unsaturated fatty acids. These findings provide the first direct evidence that the transmembrane segments of DesK are essential to sense changes in membrane fluidity and for regulating the ratio of kinase to phosphatase activities of the cytoplasmic C-terminal domain.
Chemotactic Signaling by an
Escherichia coli
CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners
Authors: Jahreis Knut , Morrison Tom B. , Garzón Andrés , Parkinson John S. 
Published: April 2004

ABSTRACT

CheA is a multidomain histidine kinase for chemotaxis in
Escherichia coli
. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheAΔP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheAΔP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheAΔP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheAΔP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.
Conditional Lethal Amber Mutations in Essential
Escherichia coli
Genes
Authors: Herring Christopher D. , Blattner Frederick R. 
Published: April 2004

ABSTRACT

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in
Escherichia coli
that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential
E. coli
genes. Amber stop codons were introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes
frr
,
gcpE, lpxC
,
map
,
murA
,
ppa
, and
rpsA
. We were unable to isolate an amber mutation in
ftsZ
. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in
gcpE
, which was bacteriostatic. The strain carrying an amber mutation in
murA
was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.
LuxS-Mediated Signaling inStreptococcus mutansIs Involved in Regulation of Acid and Oxidative Stress Tolerance and Biofilm Formation
Authors: Wen Zezhang T. , Burne Robert A. 
Published: April 2004

ABSTRACTLuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role ofluxSofStreptococcus mutansin the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation ofluxSresulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes includedffh(a signal recognition particle subunit) andbrpA(biofilm regulatory protein A). Interestingly, theluxSmutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). TheluxSmutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci,smu486andsmu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of thesmu486 smu487mutant were, in almost all cases, similar to those of theluxSmutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates thatluxS-dependent signaling plays critical roles in modulating key virulence properties ofS. mutans.
The Protease Lon and the RNA-Binding Protein Hfq Reduce Silencing of theEscherichia coli bglOperon by H-NS
Authors: Dole Sudhanshu , Klingen Yvonne , Nagarajavel V. , Schnetz Karin 
Published: April 2004

ABSTRACTThe histone-like nucleoid structuring protein H-NS represses theEscherichia coli bgloperon at two levels. H-NS binds upstream of the promoter, represses transcription initiation, and binds downstream within the coding region of the first gene, where it induces polarity of transcription elongation. Inhnsmutants, silencing of thebgloperon is completely relieved. Various screens for mutants in which silencing ofbglis reduced have yielded mutations inhnsand in genes encoding the transcription factors LeuO and BglJ. In order to identify additional factors that regulatebgl, we performed a transposon mutagenesis screen for mutants in which silencing of the operon is strengthened. This screen yielded mutants with mutations incyaA,hfq,lon, andpgi, encoding adenylate cyclase, RNA-binding protein Hfq, protease Lon, and phosphoglucose isomerase, respectively. IncyaAmutants, the cyclic AMP receptor protein-dependent promoter is presumably inactive. The specific effect of thepgimutants onbglis low. Interestingly, in thehfqandlonmutants, the downstream silencing ofbglby H-NS (i.e., the induction of polarity) is more efficient, while the silencing of the promoter by H-NS is unaffected. Furthermore, in anhnsmutant, Hfq has no significant effect and the effect of Lon is reduced. These data provide evidence that the specific repression by H-NS can (directly or indirectly) be modulated and controlled by other pleiotropic regulators.
The
pgaABCD
Locus of
Escherichia coli
Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation
Authors: Wang Xin , Preston James F. , Romeo Tony 
Published: April 2004

ABSTRACT

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of
Escherichia coli
biofilm formation suggested the involvement of an unknown adhesin. We now establish that the
pgaABCD
(formerly
ycdSRQP
) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the
pga
genes are required for optimal biofilm formation under a variety of growth conditions. A
pga
-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched β-1,6-
N
-acetyl-
d
-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The
pga
genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-β-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The
pgaABCD
operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of
E. coli
and other bacteria.
Involvement of Error-Prone DNA Polymerase IV in Stationary-Phase Mutagenesis in
Pseudomonas putida
Authors: Tegova Radi , Tover Andres , Tarassova Kairi , Tark Mariliis , Kivisaar Maia 
Published: April 2004

ABSTRACT

In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the
dinB
gene) in stationary-phase mutagenesis in
Pseudomonas putida
. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g., 1-bp deletions and different base substitutions) separately. A significant effect of Pol IV became apparent when the frequency of accumulation of 1-bp deletion mutations was compared in the
P. putida
wild-type strain and its Pol IV-defective
dinB
knockout derivative. Pol IV-dependent mutagenesis caused a remarkable increase (approximately 10-fold) in the frequency of accumulation of 1-bp deletion mutations on selective plates in wild-type
P. putida
populations starved for more than 1 week. No effect of Pol IV on the frequency of accumulation of base substitution mutations in starving
P. putida
cells was observed. The occurrence of 1-bp deletions in
P. putida
cells did not require a functional RecA protein. RecA independence of Pol IV-associated mutagenesis was also supported by data showing that transcription from the promoter of the
P. putida dinB
gene was not significantly influenced by the DNA damage-inducing agent mitomycin C. Therefore, we hypothesize that mechanisms different from the classical RecA-dependent SOS response could elevate Pol IV-dependent mutagenesis in starving
P. putida
cells.
Physiologic and Proteomic Evidence for a Role of Nitric Oxide in Biofilm Formation by
Nitrosomonas europaea
and Other Ammonia Oxidizers
Authors: Schmidt Ingo , Steenbakkers Peter J. M. , op den Camp Huub J. M. , Schmidt Katrin , Jetten Mike S. M. 
Published: April 2004

ABSTRACT

NO, a free radical gas, is the signal for
Nitrosomonas europaea
cells to switch between different growth modes. At an NO concentration of more than 30 ppm, biofilm formation by
N. europaea
was induced. NO concentrations below 5 ppm led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the proteins expressed by
N. europaea
were identified. Comparison studies of the protein patterns of motile-planktonic and attached (biofilm) cells revealed several clear differences. Eleven proteins were found to be up or down regulated. Concentrations of other compounds such as ammonium, nitrite, and oxygen as well as different temperatures and pH values had no significant effect on the growth mode of and the proteins expressed by
N. europaea
.
RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of
Corynebacterium glutamicum
Authors: Gerstmeir Robert , Cramer Annette , Dangel Petra , Schaffer Steffen , Eikmanns Bernhard J. 
Published: April 2004

ABSTRACT

The adaptation of
Corynebacterium glutamicum
to acetate as a carbon and energy source involves transcriptional regulation of the
pta-ack
operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the
aceA
and
aceB
genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as
cis
-regulatory elements for expression of the
pta-ack
operon and the
aceA
and
aceB
genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the
pta-ack
operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the
pta-ack
and the
aceA/aceB
promoter/operator regions. Directed deletion of the
ramB
gene in the genome of
C. glutamicum
resulted in mutant strain RG1. Whereas the wild type of
C. glutamicum
showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional
cat
fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of
C. glutamicum
.
Haloviruses HF1 and HF2: Evidence for a Recent and Large Recombination Event
Authors: Tang Sen-Lin , Nuttall Stewart , Dyall-Smith Mike 
Published: April 2004

ABSTRACT
Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.
Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for
Escherichia coli
RfaH both In Vivo and In Vitro
Authors: Carter Heather D. , Svetlov Vladimir , Artsimovitch Irina 
Published: April 2004

ABSTRACT

The transcriptional enhancer protein RfaH positively regulates production of virulence factors in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium via a
cis
element,
ops
. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including
Vibrio
and
Yersinia
spp. We cloned the
rfaH
genes from
Vibrio cholerae, Yersinia enterocolitica, S. enterica
serovar Typhimurium, and
Klebsiella pneumoniae
into
E. coli
expression vectors. Purified RfaH orthologs, including the most divergent one from
V. cholerae
, were readily recruited to the
E. coli
transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to
E. coli
RfaH.
V. cholerae
RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the
rfaH
genes complemented the disruption of the chromosomal copy of the
E. coli
gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the
E. coli
system.
Genome of Staphylococcal Phage K: a New Lineage of
Myoviridae
Infecting Gram-Positive Bacteria with a Low G+C Content
Authors: O'Flaherty S. , Coffey A. , Edwards R. , Meaney W. , Fitzgerald G. F. , Ross R. P. 
Published: April 2004

ABSTRACT

Phage K is a polyvalent phage of the
Myoviridae
family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from
Listeria
phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (
lys
-I1,
pol
-I2, and
pol
-I3) encoding putative endonucleases were located in the genome. Two of these (
pol
-I2 and
pol
-I3) were found to interrupt the DNA polymerase gene, while the other (
lys
-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX
2
CX
22
CX
2
C and CX
2
CX
23
CX
2
C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.