bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, Sequences, and Characterization of Two Chitinase Genes from the Antarctic
Arthrobacter
sp. Strain TAD20: Isolation and Partial Characterization of the Enzymes
Authors: Lonhienne Thierry , Mavromatis Konstantinos , Vorgias Constantin E. , Buchon Laurent , Gerday Charles , Bouriotis Vassilis 
Published: July 2002

ABSTRACT

Arthrobacter
sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell.
Arthrobacter
sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (
Ar
ChiA and
Ar
ChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in
Escherichia coli
. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of
Ar
ChiA (881 amino acids [aa]) and
Ar
ChiB (578 aa).
Ar
ChiA and
Ar
ChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of
Ar
ChiA exhibits 55% identity with a chitodextrinase from
Vibrio furnissii
. The
Ar
ChiB catalytic domain exhibits 33% identity with chitinase A of
Bacillus circulans
. The
Ar
ChiA chitin-binding domains are homologous to the chitin-binding domain of
Ar
ChiB.
Ar
ChiA and
Ar
ChiB were purified to homogeneity from the native
Arthrobacter
strain and partially characterized. Thermal unfolding of
Ar
ChiA,
Ar
ChiB, and chitinase A of
Serratia marcescens
was studied using differential scanning calorimetry.
Ar
ChiA and
Ar
ChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.
Programmed Cell Death in
Escherichia coli
: Some Antibiotics Can Trigger
mazEF
Lethality
Authors: Sat Boaz , Hazan Ronen , Fisher Tova , Khaner Hanita , Glaser Gad , Engelberg-Kulka Hanna 
Published: July 2002

ABSTRACT

The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of
Escherichia coli
, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the
E. coli mazEF
system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the
E. coli mazEF
module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.
Functional Characterization of Alanine Racemase from
Schizosaccharomyces pombe
: a Eucaryotic Counterpart to Bacterial Alanine Racemase
Authors: Uo Takuma , Yoshimura Tohru , Tanaka Naotaka , Takegawa Kaoru , Esaki Nobuyoshi 
Published: July 2002

ABSTRACT

Schizosaccharomyces pombe
has an open reading frame, which we named
alr1
+
, encoding a putative protein similar to bacterial alanine racemase. We cloned the
alr1
+
gene in
Escherichia coli
and purified the gene product (Alr1p), with an
M
r
of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent

K
m

and
V
max
values as follows: for
l
-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for
d
-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but
l
-serine and
l
-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of
l
-alanine, respectively.
S. pombe
uses
d
-alanine as a sole nitrogen source, but deletion of the
alr1
+
gene resulted in retarded growth on the same medium. This indicates that
S. pombe
has catabolic pathways for both enantiomers of alanine and that the pathway for
l
-alanine coupled with racemization plays a major role in the catabolism of
d
-alanine.
Saccharomyces cerevisiae
differs markedly from
S. pombe
:
S. cerevisiae
uses
l
-alanine but not
d
-alanine as a sole nitrogen source. Moreover,
d
-alanine is toxic to
S. cerevisiae
. However, heterologous expression of the
alr1
+
gene enabled
S. cerevisiae
to grow efficiently on
d
-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of
d
-alanine.
Cloning and Characterization of the Gene Cluster for Palatinose Metabolism from the Phytopathogenic BacteriumErwinia rhapontici
Authors: Börnke Frederik , Hajirezaei Mohammad , Sonnewald Uwe 
Published: July 2002

ABSTRACTErwinia rhaponticiis able to convert sucrose into isomaltulose (palatinose, 6-O-α-d-glucopyranosyl-d-fructose) and trehalulose (1-O-α-d-glucopyranosyl-d-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes fromErwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. Thepalgene products ofErwinia rhaponticiwere shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. ThepalE, palF, palG, palH, palK, palQ, andpalZgenes were oriented divergently with respect to thepalRandpalIgenes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of thepalIgene and thepalEFGHKQZgenes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression ofpalIandpalQinEscherichia colirevealed sucrose isomerase and palatinase activity, respectively.
Functional Analysis of the
Erwinia herbicola tutB
Gene and Its Product
Authors: Katayama Takane , Suzuki Hideyuki , Koyanagi Takashi , Kumagai Hidehiko 
Published: July 2002

ABSTRACT

The
tutB
gene, which lies just downstream of
tpl
, has been cloned from
Erwinia herbicola
, and its product was analyzed. Despite its high sequence similarity to tryptophan transporters, TutB was found to be a tyrosine-specific transporter. Tryptophan acted as a competitive inhibitor of tyrosine transport. Unlike the tryptophanase operon, the
tpl
and
tutB
genes do not constitute an operon.
Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes
Authors: Murata Takahiro , Ohnishi Makoto , Ara Takeshi , Kaneko Jun , Han Chang-Gyun , Li Yong Fang , Takashima Kayoko , Nojima Hideaki , Nakayama Keisuke , Kaji Akira , Kamio Yoshiyuki , Miki Takeyoshi , Mori Hirotada , Ohtsubo Eiichi , Terawaki Yoshiro , Hayashi Tetsuya 
Published: July 2002

ABSTRACTRts1, a large conjugative plasmid originally isolated fromProteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence oftus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.
Quorum-Sensing Signals and Quorum-Sensing Genes in
Burkholderia vietnamiensis
Authors: Conway Barbara-Ann , Greenberg E. P. 
Published: July 2002

ABSTRACT

Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many
Proteobacteria
including a clinical isolate of
Burkholderia cepacia
. The
B. cepacia
isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of
Burkholderia vietnamiensis
. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in
B. vietnamiensis
cultures reached concentrations in excess of 20 μM. We isolated a
B. vietnamiensis
DNA fragment containing a gene for the synthesis of decanoyl-HSL (
bviI
) and an open reading frame that codes for a putative signal receptor (
bviR
). A
B. vietnamiensis bviI
mutant did not produce detectable levels of decanoyl-HSL.
Direct Identification of Bacteria from Positive Blood Cultures by Amplification and Sequencing of the 16S rRNA Gene: Evaluation of BACTEC 9240 Instrument True- Positive and False-Positive Results
Authors: Qian Qinfang , Tang Yi-Wei , Kolbert Christopher P. , Torgerson Catherine A. , Hughes John G. , Vetter Emily A. , Harmsen W. Scott , Montgomery Stacy O. , Cockerill Franklin R. , Persing David H. 
Published: July 2002

ABSTRACT

In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (
n
= 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (
n
= 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (
P
= 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.
Low Concentrations of Mupirocin in the Pharynx following Intranasal Application May Contribute to Mupirocin Resistance in Methicillin-Resistant
Staphylococcus aureus
Authors: Watanabe Hiroshi , Masaki Hironori , Asoh Norichika , Watanabe Kiwao , Oishi Kazunori , Kobayashi Shinobu , Sato Akiyoshi , Sugita Rinya , Nagatake Tsuyoshi 
Published: July 2002

ABSTRACT

We describe a patient with methicillin-resistant
Staphylococcus aureus
(MRSA) colonizing the pharynx. The MIC of mupirocin was 0.25 μg/ml before treatment and increased after treatment to 8 μg/ml. Using pulsed-field gel electrophoresis, we confirmed that the genotypes of MRSA that colonized the pharynx before and after the use of mupirocin were identical. We measured the delivery of mupirocin to the pharynx in three normal volunteers and two patients. Low concentrations of mupirocin were present in the pharynx in all cases 10 min to 3 days after intranasal application. Our data suggested that low concentrations of the drug in the pharynx after intranasal application of mupirocin ointment might explain the selection of mupirocin resistance in MRSA.
Prevalence of Anti-Human Papillomavirus Type 16, 18, 31, and 58 Virus-Like Particles in Women in the General Population and in Prostitutes
Authors: Touzé Antoine , de Sanjosé Silvia , Coursaget Pierre , Almirall M. Rosa , Palacio Virgilio , Meijer Chris J. L. M. , Kornegay Janet , Bosch F. Xavier 
Published: July 2002

ABSTRACT
Genital human papillomavirus (HPV) infection is sexually transmitted. The aim of the study was to characterize serological responses to HPV types 16, 18, 31, and 58 by exploring type-specific virus-like particles (VLPs) in two groups of women with very distinct sexual behaviors. Anti-VLP antibodies for types 16, 18, 31, and 58 and HPV DNA in cervical cells were investigated with 177 prostitutes and 283 age-matched controls from the female general population in Spain. Anti-VLP positivity increased with number of lifetime sexual partners in women from the general population, and no seroresponse was found in virgins. However, in prostitutes HPV infection was characterized by higher multireactivity to three or four VLPs (25%) than the general population (3%) and by a more frequent antibody response to HPV-58 than in the general population. About 75% of the women seropositive for type 58 had been born in a Latin American country. Seroprevalence of HPV and cervical HPV DNA in prostitutes were 14 and 10 times higher than observed in women in the general population (prevalence odds ratio [POR] of HPV seropositivity, 14.04 [95%; CI = 8.4 to 23.6] and POR for HPV DNA, 10.4 [95% CI = 3.9 to 27.6). Our results indicate that prostitutes are at an increased risk of oncogenic HPV infections, and they confirm the validity of anti-VLPs as markers of present or past HPV infection, that the number of sexual partners is the major determinant in acquisition of oncogenic HPV, and that anti-VLPs could be used as a marker of repeated infection in prostitutes.
Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis
Authors: Allard Annika , Albinsson Bo , Wadell Göran 
Published: July 2002

ABSTRACT
We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.
Improved, Low-Cost Selective Culture Medium for
Actinobacillus actinomycetemcomitans
Authors: Alsina M. , Olle E. , Frias J. 
Published: July 2002

ABSTRACT

Actinobacillus actinomycetemcomitans
is considered to be one of the major oral putative pathogens, especially in cases of juvenile periodontitis. This microorganism requires nutritionally complex media for growth, and therefore the media for its primary isolation usually include blood agar or serum in their base. In this study we present a new medium, Dentaid-1, which improves the detection of
A. actinomycetemcomitans
in periodontal samples. In its composition, blood and serum have been omitted, hence reducing its cost and making it a more restrictive medium against the growth of other microorganisms with high nutritional requirements. The growth yields of pure cultures of the bacteria on Dentaid-1 were comparable to those on nonselective blood agar. Moreover, clinical efficacy was evaluated in subgingival samples from 77 subjects with adult periodontitis. Dentaid-1 detected
A. actinomycetemcomitans
in 24 subjects, while a previously described tryptic soy-serum-bacitracin-vancomycin agar detected the microorganism in only 19 subjects (79.1%). Dentaid-1 is a low-cost, noninhibitory formula for the improved diagnosis and monitoring of patients subgingivally infected by this important oral putative pathogen.
Evaluation of a Medium (STGG) for Transport and Optimal Recovery of
Streptococcus pneumoniae
from Nasopharyngeal Secretions Collected during Field Studies
Authors: O'Brien Katherine L. , Bronsdon Melinda A. , Dagan Ron , Yagupsky Pablo , Janco Jacob , Elliott John , Whitney Cynthia G. , Yang Yong-Hong , Robinson Lisa-Gaye E. , Schwartz Benjamin , Carlone George M. 
Published: July 2002

ABSTRACT

Field studies of
Streptococcus pneumoniae
(pneumococci) nasopharyngeal (NP) colonization are hampered by the need to directly plate specimens in order to ensure isolate viability. A medium containing skim milk, tryptone, glucose, and glycerin (STGG) has been used to transport and store NP material, but its ability to preserve pneumococci has not been evaluated. Our objective was to qualitatively and semiquantitatively evaluate the ability of STGG to preserve pneumococci in NP secretions. Entwined duplicate calcium alginate NP swab samples were obtained from children. One swab was plated directly onto a gentamicin blood agar plate; the other was placed in STGG. Growth from the directly plated specimen was compared with growth from an STGG aliquot immediately cultured or stored at −70°C for 9 weeks, −20°C for 9 weeks, or 4°C for 5 days. Of 186 specimens, 96 (52%) were positive for pneumococci from the direct plating; 94 (98%) of these were positive from the fresh STGG specimen. Pneumococci were recovered from all 38 positive specimens frozen at −70°C, all 18 positive specimens frozen at −20°C, and 18 of 20 positive specimens stored at 4°C. Recovery of pneumococci after storage of NP material in STGG medium at −70°C is at least as good as that from direct plating. Storage at −20°C is also acceptable. Storage at 4°C for 5 days is not ideal.
Performance Assessment of New Multiplex Probe Assay for Identification of Mycobacteria
Authors: Tortoli Enrico , Nanetti Anna , Piersimoni Claudio , Cichero Paola , Farina Claudio , Mucignat Giorgio , Scarparo Claudio , Bartolini Laura , Valentini Roberta , Nista Domenico , Gesu Giampietro , Tosi Cristiana Passerini , Crovatto Marina , Brusarosco Giuliana 
Published: July 2002

ABSTRACT

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of
Mycobacterium tuberculosis
complex,
Mycobacterium kansasii
,
Mycobacterium xenopi
,
Mycobacterium gordonae
, the species of the
Mycobacterium avium
complex (MAC),
Mycobacterium scrofulaceum
, and
Mycobacterium chelonae
was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus
Mycobacterium
, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as
Mycobacterium intracellulare
by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species
M. kansasii
and
M. chelonae
, while the declared overlapping reactivity of probe 4 with some
M. kansasii
and
Mycobacterium gastri
organisms and of probe 9 with MAC,
Mycobacterium haemophilum
, and
Mycobacterium malmoense
, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.