bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integrative module of the high‐pathogenicity island of Yersinia
Authors: Rakin A. , Noelting C. , Schropp P. , Heesemann J. 
Published: March 2003

The high‐pathogenicity island of Yersinia pestis (Yps HPI) encodes virulence‐associated genes involved in siderophore yersiniabactin‐mediated iron uptake. The Yps HPI contains a P4‐type integrase (Int‐HPI), associated with the asn‐tRNA locus, and is flanked by 17 bp direct repeats. We constructed a minimal integrative module of the pathogenicity island carrying the reconstituted 266 bp attP (POP′) attachment site derived from putative attR and attL junctions of the Yps HPI and the functional int‐HPI gene from Y. pestis KUMA. The attP–int‐HPI module recombined efficiently, site specifically and RecA independently with the bacterial attB site present either in the chromosome (asn‐tDNA) or on a plasmid, with no preference for a certain asn‐tRNA gene. The excision of the integrated suicide plasmid carrying the integrative module, on the other hand, was a rare event and could be demonstrated only by polymerase chain reaction. Analysis of the 5′ terminus of the transcript for int‐HPI revealed that the integration of attP–int‐HPI was coupled with the replacement of the endogenous int‐HPI promoter, localized in the P′ part of the attP site, by the adjacent asn‐tRNA promoter. These results suggest that two alternative promoters control integration and excision of the HPI by its integrase.
Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella
Authors: Figueroa‐Bossi Nara , Uzzau Sergio , Maloriol Danièla , Bossi Lionello 
Published: March 2003

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy‐1 and Gifsy‐2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy‐2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III‐translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy‐1 and Gifsy‐2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels‐1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy‐3, which contains the phoP/phoQ‐activated pagJ gene and the gene for the secreted leucine‐rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEΦ. Most phage‐associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.
Molecular cloning and genetic analysis of an indole‐diterpene gene cluster from Penicillium paxilli
Authors: Young Carolyn , McMillan Lisa , Telfer Emily , Scott Barry 
Published: March 2003

The indole‐diterpene paxilline is a potent tremorgenic mammalian mycotoxin and a known inhibitor of maxi‐K ion channels. The gene cluster responsible for paxilline biosynthesis in Penicillium paxilli was identified by mapping four large plasmid‐induced chromosome deletions. The cluster is predicted to lie within a 50 kb region of chromosome Va and to contain 17 genes, including a geranylgeranyl pyrophosphate (GGPP) synthase (paxG), two FAD‐dependent monooxygenases (paxM and N), two cytochrome P450 monooxygenases (paxP and Q), a dimethylallyltryptophan (DMAT) synthase (paxD) and two possible transcription factors (paxR and paxS), which contain a Zn(II)2Cys6 DNA‐binding motif. Targeted replacement of paxG confirmed that it is essential for paxilline biosynthesis but dispensable for growth. The GGPP for primary metabolism is predicted to be provided by a second GGPP synthase (ggs1) that was cloned, sequenced and mapped to chromosome IV. Semi‐quantitative reverse transcriptase–polymerase chain reaction analysis demonstrated that the expression of paxG, paxM and paxP in submerged liquid cultures of P. paxilli increased dramatically with the onset of paxilline biosynthesis. In contrast, the expression of β‐tubulin (tub2) and ggs1 was not induced. This is the first description of the molecular cloning and genetic analysis of an indole‐diterpene gene cluster.
Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus
Authors: Quardokus Ellen M. , Din Neena , Brun Yves V. 
Published: March 2003

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid‐cell early in the cell cycle. In an effort to understand whether Z‐ring formation and cell constriction are driven solely by the cell cycle‐regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z‐ring formation; Z‐ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid‐cell constriction. These results suggest that the timing and placement of Z‐rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.
Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551
Authors: Kunnimalaiyaan Muthusamy , Stevenson David M. , Zhou Yansheng , Vary Patricia S. 
Published: March 2003

An 18 633 bp region containing the replicon from the ≈ 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non‐essential plasmid DNA has implications for systematics and evolutionary biology.
Mismatch repair and the regulation of phase variation in Neisseria meningitidis
Authors: Richardson A. R. , Stojiljkovic I. 
Published: March 2003

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from ≈ 10−6 to 10−2 cfu−1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500‐ and 250‐fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20‐ to 30‐fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase‐variable genes without an accompanied increased accumulation of spontaneous mutations.
Replication restart in UV‐irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins
Authors: Rangarajan Savithri , Woodgate Roger , Goodman Myron F. 
Published: March 2003

SummaryIn Escherichia coli, UV‐irradiated cells resume DNA synthesis after a transient
inhibition by a process called replication restart. To elucidate the role of several
key proteins involved in this process, we have analysed the time dependence of replication
restart in strains carrying a combination of mutations in lexA, recA,
polB (pol II), umuDC (pol V), priA, dnaC, recF,
recO or recR. We find that both pol II and the origin‐independent primosome‐assembling
function of PriA are essential for the immediate recovery of DNA synthesis after
UV irradiation. In their absence, translesion replication or ‘replication
readthrough’ occurs ≈ 50 min after UV and is pol V‐dependent. In
a wild‐type, lexA+ background, mutations in recF,
recO or recR block both pathways. Similar results were obtained with
a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def)
recR strains, although unable to facilitate PriA‐pol II‐dependent restart,
were able to perform pol V‐dependent readthrough. The defects in restart attributed
to mutations in recF, recO or recR were suppressed in a recA730
lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest
that in a wild‐type background, RecF, O and R are important for the induction of
the SOS response and the formation of RecA*‐dependent recombination intermediates
necessary for PriA/Pol II‐dependent replication restart. In con‐trast, only RecF
is required for the activation of RecA that leads to the formation of pol V (UmuD′2C) and facilitates replication readthrough.
Screening for the target gene of cyanobacterial cAMP receptor protein SYCRP1
Authors: Yoshimura Hidehisa , Yanagisawa Shuichi , Kanehisa Minoru , Ohmori Masayuki 
Published: March 2003

SummaryThe target genes for SYCRP1, a cyanobacterial cAMP receptor protein, were surveyed using a DNA micro‐array method. Total RNAs were extracted from a wild‐type strain and a sycrp1 disruptant of Synechocystis sp. PCC 6803, and the respective
gene expression levels were compared. The expression levels of six genes (slr1667,
slr1668, slr2015, slr2016, slr2017 and slr2018) were clearly decreased by the disruption
of the sycrp1 gene. The data suggest that slr1667 and slr1668 constitute one
operon and the other four genes constitute another operon. Transcription start points
for the first genes of these putative operons, which are slr1667 and slr2015, were
determined by primer extension experiments. Gel mobility shift assays and DNase I
footprint analyses were carried out to explore the binding of SYCRP1 to the putative
promoter regions of slr1667 and slr2015. SYCRP1 bound to the specific site in the
5′ upstream region of slr1667 from positions –170 to –155
relative to the transcription start point, while it did not bind to the 5′ upstream region of slr2015. It was concluded that SYCRP1 regulates the expression of the slr1667 gene directly by binding to a specific site in its promoter.
A distinctive single‐stranded DNA‐binding protein from the Archaeon Sulfolobus solfataricus
Authors: Haseltine Cynthia A. , Kowalczykowski Stephen C. 
Published: March 2003

SummarySingle‐stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryar‐chaeal counterparts. Rather than comprising four DNA‐binding domains and a zinc‐finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus

SSB protein (SsoSSB) has a single DNA‐binding domain in a polypeptide of just 148

amino acids with a eubacterial‐like acidic C‐terminus. SsoSSB protein was purified

to homogeneity and found to form tetramers in solution, suggesting a quaternary structure

analogous to that of E. coli SSB protein, despite possessing DNA‐binding domains

more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive

binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately

five nucleotides (nt) per monomer. Additionally, the protein is functional both in

vitro and in vivo, stimulating RecA protein‐mediated DNA strand‐exchange

and rescuing the ssb‐1 lethal mutation of E. coli respectively. We dis‐cuss possible evolutionary relationships amongst the various members of the SSB/RPA family.
Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes
Authors: Baron Christian , O´Callaghan David , Lanka Erich 
Published: March 2003

SummaryType IV secretion systems (TFSS) mediate secretion or direct cell‐to‐cell transfer of virulence factors (proteins or protein–DNA complexes) from many Gram‐negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella
tribocorum, Bordetella pertussis, Brucella suis, Helicobacter
pylori, Legionella pneumophila and Rickettsia prowazekii, into
eukaryotic cells. Bacterial conjugation is also classified as a TFSS‐like process
mediating the spread of broad‐host‐range plasmids between Gram‐negative bacteria
such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical,
cell biological and structural biology experiments led to significant progress in
the understanding of several aspects of TFSS processes. X‐ray crystallography revealed
that homologues of the A. tumefaciens inner membrane‐associated proteins VirB11
and VirD4 from H. pylori and R388, respectively, may form channels for substrate
translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell
biological experiments revealed interactions between components of the periplasmic
core components VirB8, VirB9 and VirB10, which may form the translocation channel.
Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that
the periplasmic translocation route of the pertussis toxin from B. pertussis
may be more generally valid than previously anticipated. Secretion and modification
of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS‐carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell–cell interactions.
Involvement of caspase‐3‐like protein in rapid cell death of Xanthomonas
Authors: Gautam Satyendra , Sharma Arun 
Published: March 2003

Summary Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the aetiological agent of bacterial pustule disease of soybean, as well as some other strains of Xanthomonas including X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961, exhibited post‐exponential rapid cell death (RCD) in Luria–Bertani (LB) medium. RCD was not displayed by Xanthomonas strains while growing in starch medium. Addition of starch to LB culture of XcgAM2 at any point of incubation during the exponential growth was found to arrest the onset of RCD. RCD in this organism was found to be associated with the synthesis of an endogenous enzyme similar to human caspase‐3, a known marker of apoptosis in eukaryotes. On sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS‐PAGE) the XcgAM2 caspase appeared to run along a 55 kDa protein molecular weight marker. The caspase‐3‐like protein was detected in all Xanthomonas strains tested. RCD was not detected in Escherichia coli cultures in LB medium. The caspase‐3‐like enzyme activity or pro‐tein was also found to be absent in this bacterium. Caspase‐3‐like protein or Xanthomonas caspase was detected only in the cells of XcgAM2 growing in LB medium and not in those growing in starch medium. The Xanthomonas caspase protein appeared in cells at around 4 h of incubation, and peaked at around 24 h, before finally disappearing at around 54 h of incubation. However, caspase enzyme activity was detected only 12–13 h after incubation and peaked around 18–20 h. Addition of starch at the beginning or during the period of exponential growth in LB cultures of XcgAM2 terminated the synthesis of this protein. It is presumed that starch acted as the repressor of biosynthesis of the Xanthomonas caspase, thereby preventing the organism from undergoing RCD. The cells undergoing RCD also displayed the other markers of eukaryotic apoptosis. These included binding of annexin V to plasma membrane of cells undergoing RCD and the presence of nicked DNA in culture supernatant as evidenced by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labelling) assay. Caspase‐negative mutants of XcgAM2 did not display post‐exponential RCD. The importance of RCD in Xanthomonas life cycle is not yet clear, however the phenomenon appears to have similarities with eukaryotic apoptosis.
The C‐terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga
Authors: Molmeret Maëlle , Alli O. A. Terry , Radulic Marina , Susa Milorad , Doric Miljenko , Kwaik Yousef Abu 
Published: March 2003

SummaryWe have shown previously that the five rib (release of intracellular
bacteria) mutants of Legionella pneumophila are competent for intracellular
replication but defective in pore formation‐mediated cytolysis and egress from protozoan
and mammalian cells. The rib phenotype results from a point mutation (deletion)
ΔG544 in icmT that is predicted to result in the expression
of a protein truncated by 32 amino acids from the C‐terminus. In contrast to the
rib mutants that are capable of intracellular replication, an icmT
null mutant was completely defective in intracellular replication within mammalian
and protozoan cells, in addition to its defect in pore formation‐mediated cytolysis.
The icmT wild‐type allele complemented the icmT null mutant for both
defects of intracellular replication and pore formation‐mediated cytolysis and egress
from mammalian cells. In contrast, the icmTΔG544 allele
complemented the icmT null mutant for intracellular growth, but not for the
pore‐forming activity. Consistent with their defect in pore formation‐mediated cytotoxicity
in vitro, both mutants failed to cause pulmonary inflammation in A/J mice.
Interestingly, the rib mutant was severely defective in intracellular growth
within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy
confirmed that the rib mutant and the icmT null mutant were severely
and completely defective, respectively, in intracellular growth in A. polyphaga,
and the respective defects correlated with fusion of the bacterial phagosomes to
lysosomes. Taken together, the data showed that the C‐terminus domain of IcmT is
essential for the pore‐forming activity and is required for intracellular trafficking
and replication within A. polyphaga, but not within mammalian cells.
Bacterial resistance evolution by recruitment of super‐integron gene cassettes
Authors: Rowe‐Magnus Dean A. , Guerout Anne‐Marie , Mazel Didier 
Published: March 2003

SummaryThe capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram‐negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super‐integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring

MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae,

a species known to harbour a SI. We show that MRIs can randomly recruit genes directly

from the cache of SI cassettes. By applying a selective constraint for the development

of antibiotic resistance, we demonstrate bacterial resistance evolution through the

recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase

gene from the V. cholerae SI and its precise insertion into the MRI. The resulting

resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron‐mediated recombination events.