bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Sulfur-Regulated Arylsulfatase Gene Cluster of
Pseudomonas aeruginosa
, a New Member of the
cys
Regulon
Authors: Hummerjohann Jörg , Laudenbach Sascha , Rétey Julia , Leisinger Thomas , Kertesz Michael A. 
Published: July 2002

ABSTRACT

A gene cluster upstream of the arylsulfatase gene (
atsA
) in
Pseudomonas aeruginosa
was characterized and found to encode a putative ABC-type transporter, AtsRBC. Mutants with insertions in the
atsR
or
atsB
gene were unable to grow with hexyl-, octyl-, or nitrocatecholsulfate, although they grew normally with other sulfur sources, such as sulfate, methionine, and aliphatic sulfonates. AtsRBC therefore constitutes a general sulfate ester transport system, and desulfurization of aromatic and medium-chain-length aliphatic sulfate esters occurs in the cytoplasm. Expression of the
atsR
and
atsBCA
genes was repressed during growth with sulfate, cysteine, or thiocyanate. No expression of these genes was observed in the
cysB
mutant PAO-CB, and the
ats
genes therefore constitute an extension of the
cys
regulon in this species.
Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in
Synechocystis
sp. Strain PCC 6803
Authors: Ermakova-Gerdes Svetlana , Yu Zhenbao , Vermaas Wim 
Published: July 2002

ABSTRACT

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium
Synechocystis
sp. strain PCC 6803, we randomly mutagenized a region of
psbDI
(coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a
Synechocystis
sp. strain PCC 6803 mutant that lacks both
psbD
genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q
A
-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in
Synechocystis
sp. strain PCC 6803 was developed to facilitate genetic analysis of
psbDI
mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in
Synechocystis
sp. strain PCC 6803 than was generally assumed.
Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium
Acidithiobacillus caldus
and Analysis of the Plasmid Replicon
Authors: Gardner Murray N. , Deane Shelly M. , Rawlings Douglas E. 
Published: July 2002

ABSTRACT

A moderately thermophilic (45 to 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic
Acidithiobacillus caldus
strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the
A. caldus
cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in
Escherichia coli
. Autonomous replication was also demonstrated in
Pseudomonas putida
and
Agrobacterium tumefaciens
LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14
oriV
region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both
E. coli,
and
A. caldus
. The
rep
gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's
oriV
regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in
trans
. Two smaller open reading frames were found between the
repB
and
repA
genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.
Identification of the Outer Membrane Porin of
Thermus thermophilus
HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance
Authors: Maier Elke , Polleichtner Georg , Boeck Birgit , Schinzel Reinhard , Benz Roland 
Published: July 2002

ABSTRACT

The outer membrane of the thermophilic bacterium
Thermus thermophilus
was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.
Twin-Arginine Translocation Pathway in
Streptomyces lividans
Authors: Schaerlaekens Kristien , Schierová Michaela , Lammertyn Elke , Geukens Nick , Anné Jozef , Van Mellaert Lieve 
Published: July 2002

ABSTRACT

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in
Streptomyces lividans
, a gram-positive organism with a high secretion capacity. The presence of one
tatC
and two
hcf106
homologs in the
S. lividans
genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the
S. lividans
secretome. To demonstrate its functionality, a
tatC
deletion mutant was constructed. This mutation impaired the translocation of the
Streptomyces antibioticus
tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in
Escherichia coli
, could be translocated in wild-type
S. lividans
but not in the
tatC
mutant. In contrast, the secretion of the Sec-dependent
S. lividans
subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in
Streptomyces
spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the
E. coli
TorA signal peptide can direct Tat-dependent transport in
S. lividans
.
Alternative Translation Initiation Produces a Short Form of a Spore Coat Protein in
Bacillus subtilis
Authors: Ozin Amanda J. , Costa Teresa , Henriques Adriano O. , Moran Charles P. 
Published: July 2002

ABSTRACT

During endospore formation in
Bacillus subtilis,
over two dozen polypeptides are localized to the developing spore and coordinately assembled into a thick multilayered structure called the spore coat. Assembly of the coat is initiated by the expression of morphogenetic proteins SpoIVA, CotE, and SpoVID. These morphogenetic proteins appear to guide the assembly of other proteins into the spore coat. For example, SpoVID forms a complex with the SafA protein, which is incorporated into the coat during the early stages of development. At least two forms of SafA are found in the mature spore coat: a full-length form and a shorter form (SafA-C
30
) that begins with a methionine encoded by codon 164 of
safA
. In this study, we present evidence that the expression of SafA-C
30
arises from translation initiation at codon 164. We found only a single transcript driving expression of SafA. A stop codon engineered just upstream of a predicted ribosome-binding site near codon M164 abolished formation of full-length SafA, but not SafA-C
30
. The same effect was observed with an alanine substitution at codon 1 of SafA. Accumulation of SafA-C
30
was blocked by substitution of an alanine codon at codon 164, but not by a substitution at a nearby methionine at codon 161. We found that overproduction of SafA-C
30
interfered with the activation of late mother cell-specific transcription and caused a strong sporulation block.
Alcohol-Induced Delay of Viability Loss in Stationary-Phase Cultures of
Escherichia coli
Authors: Vulić Marin , Kolter Roberto 
Published: July 2002

ABSTRACT

During prolonged incubation in stationary phase
Escherichia coli
undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain
n
-alcohols within a specific time window in stationary phase also prevents viability loss, in an
rpoS
-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of σ
s
such as cell density signals and sensing the nutrient content of the medium.
Definition of the Mycobacterial SOS Box and Use To Identify LexA-Regulated Genes inMycobacterium tuberculosis
Authors: Davis Elaine O. , Dullaghan Edith M. , Rand Lucinda 
Published: July 2002

ABSTRACTThe bases of the mycobacterial SOS box important for LexA binding were determined by replacing each base with every other and examining the effect on the induction of a reporter gene following DNA damage. This analysis revealed that the SOS box was longer than originally thought by 2 bp in each half of the palindromic site. A search of theMycobacterium tuberculosisgenome sequence with the new consensus, TCGAAC(N)4GTTCGA, identified 4 sites which were perfect matches and 12 sites with a single mismatch which were predicted to bind LexA. Genes which could potentially be regulated by these SOS boxes were ascertained from their positions relative to the sites. Examination of expression data for these genes following DNA damage identified 12 new genes which are most likely regulated by LexA as well as the knownM. tuberculosisDNA damage-inducible genesrecA,lexA, andruvC. Of these 12 genes, only 2 have a predicted function:dnaE2, a component of DNA polymerase III, andlinB, which is similar to 1,3,4,6-tetrachloro-1,4-cylcohexadiene hydrolase. Curiously, of the remaining 10 genes predicted to be LexA regulated, 7 are members of theM. tuberculosis13E12 repeat family, which has some of the characteristics of mobile elements.
Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of
Mycobacterium tuberculosis
in Mexico
Authors: Banaiee N. , Bobadilla-del-Valle M. , Bardarov S. , Riska P. F. , Small P. M. , Ponce-de-Leon A. , Jacobs W. R. , Hatfull G. F. , Sifuentes-Osornio J. 
Published: July 2002

ABSTRACT

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of
Mycobacterium tuberculosis
was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of
p
-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) was used for selective identification of
M. tuberculosis
complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.
Rapid Real-Time PCR for Determination of Penicillin Susceptibility in Pneumococcal Meningitis, Including Culture-Negative Cases
Authors: Kearns Angela M. , Graham Clive , Burdess Doreen , Heatherington Jane , Freeman Roger 
Published: July 2002

ABSTRACTA novel real-time PCR-hybridization assay was developed for the rapid (<1 h) detection of penicillin susceptibility inStreptococcus pneumoniae. When applied to 24 pneumococcal DNA-positive cerebrospinal fluid extracts, penicillin-sensitiveS. pneumoniaewas detected in all instances. Real-time PCR proved more sensitive than culture, microscopy, or antigen detection and provided susceptibility data even in culture-negative cases.
Comparison of the ESP and BACTEC Systems for Testing Susceptibilities of
Mycobacterium tuberculosis
Complex Isolates to Pyrazinamide
Authors: LaBombardi Vincent J. 
Published: July 2002

ABSTRACT

Fifty isolates of
Mycobacterium tuberculosis
complex were tested for susceptibility to pyrazinamide (PZA) by the ESP (Trek Diagnostic Systems, Westlake, Ohio) and BACTEC (BD Biosciences, Sparks, Md.) test systems. Initial results showed concordance for 48 of the isolates. On retest, the two discordant isolates were resolved in favor of the ESP system. The ESP Myco susceptibility test system generates rapid, reliable PZA test results.
Generation and Characterization of a Hepatitis C Virus NS3 Protease-Dependent Bovine Viral Diarrhea Virus
Authors: Lai Vicky C. H. , Zhong Weidong , Skelton Angela , Ingravallo Paul , Vassilev Venteislav , Donis Ruben O. , Hong Zhi , Lau Johnson Y. N. 
Published: July 2002

ABSTRACT

Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P
H/B
(wild-type HCV NS3 protease) and P
H/B(S139A)
(mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P
H/B
clone yielded viable viruses, whereas the mutant clone, P
H/B(S139A)
, failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV−Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV−Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.
Innate Immune Response of the Human Host to Exposure with Herpes Simplex Virus Type 1: In Vitro Control of the Virus Infection by Enhanced Natural Killer Activity via Interleukin-15 Induction
Authors: Ahmad Ali , Sharif-Askari Ehsan , Fawaz Lama , Menezes José 
Published: July 2002

ABSTRACTInfections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-γ], tumor necrosis factor alpha, or IFN-α). Anti-CD122 antibodies which block signaling through IL-2 receptor β chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.
Lentivirus Infection in the Brain Induces Matrix Metalloproteinase Expression: Role of Envelope Diversity
Authors: Johnston J. B. , Jiang Y. , van Marle G. , Mayne M. B. , Ni W. , Holden J. , McArthur J. C. , Power C. 
Published: July 2002

ABSTRACTInfection of the brain by lentiviruses, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV), causes inflammation and results in neurodegeneration. Molecular diversity within the lentivirus envelope gene has been implicated in the regulation of cell tropism and the host response to infection. Here, we examine the hypothesis that envelope sequence diversity modulates the expression of host molecules implicated in lentivirus-induced brain disease, including matrix metalloproteinases (MMP) and related transcription factors. Infection of primary macrophages by chimeric HIV clones containing brain-derived envelope fragments from patients with HIV-associated dementia (HAD) or nondemented AIDS patients (HIV-ND) showed that MMP-2 and -9 levels in conditioned media were significantly higher for the HAD clones. Similarly, STAT-1 and JAK-1 levels were higher in macrophages infected by HAD clones. Infections of primary feline macrophages by the neurovirulent FIV strain (V1CSF), the less neurovirulent strain (Petaluma), and a chimera containing the V1CSF envelope in a Petaluma background (FIV-Ch) revealed that MMP-2 and -9 levels were significantly higher in conditioned media from V1CSF- and FIV-Ch-infected macrophages, which was associated with increased intracellular STAT-1 and JAK-1 levels. The STAT-1 inhibitor fludarabine significantly reduced MMP-2 expression, but not MMP-9 expression, in FIV-infected macrophages. Analysis of MMP mRNA and protein levels in brain samples from HIV-infected persons or FIV-infected cats showed that MMP-2 and -9 levels were significantly increased in lentivirus-infected brains compared to those of uninfected controls. Elevated MMP expression was accompanied by significant increases in STAT-1 and JAK-1 mRNA and protein levels in the same brain samples. The present findings indicate that two lentiviruses, HIV and FIV, have common mechanisms of MMP-2 and -9 induction, which is modulated in part by envelope sequence diversity and the STAT-1/JAK-1 signaling pathway.
Transgenic or Plant Expression Vector-Mediated Recombination ofPlum Pox Virus
Authors: Varrelmann Mark , Palkovics Laszlo , Maiss Edgar 
Published: July 2002

ABSTRACTDifferent mutants of an infectious full-length clone (p35PPV-NAT) ofPlum pox virus(PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region ofZucchini yellow mosaic virusandPotato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections inNicotiana benthamianaplants. After passages in different transgenicN. benthamianaplants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3′-nontranslated region (3′-NTR) (plant line 17.27.4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3′-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector ofPotato virus Xexpressing the intact PPV-NAT CP gene transiently in nontransgenicN. benthamianaplants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.