bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Submitochondrial Localization of the α Subunit of the Mitochondrial Processing Peptidase from the Aquatic Fungus
Blastocladiella emersonii
Authors: Rocha Cíntia Renata Costa , Gomes Suely Lopes 
Published: December 2019

ABSTRACT

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus
Blastocladiella emersonii
, the gene encoding the α subunit of the mitochondrial processing peptidase (α-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted α-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the
B. emersonii
α-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration,
B. emersonii
α-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of
B. emersonii
α-MPP and β-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the
bc1
complex, as described for plants.
Characterization of the Serogroup O11 O-Antigen Locus of
Pseudomonas aeruginosa
PA103
Authors: Dean C. R. , Franklund C. V. , Retief J. D. , Coyne M. J. , Hatano K. , Evans D. J. , Pier G. B. , Goldberg J. B. 
Published: December 2019

ABSTRACT

We previously cloned a genomic DNA fragment from the serogroup O11
Pseudomonas aeruginosa
strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant
Escherichia coli
and
Salmonella
. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as
wzz

Pa
O111

(
wzz
from
P. aeruginosa
serogroup O11),
wzx

Pa
O11

,
wbjA
,
wzy

Pa
O11

,
wbjB
to
wbjF
,
wbpL
O11
and
wbpM
O11
(
wbpL
and
wbpM
from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of
wbpM
O11
has a markedly reduced G+C content relative to the chromosomal average. Wzy
PaO11
shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in
wzy

Pa
O11

in
P. aeruginosa
PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in
Staphylococcus aureus
. There is nearly total identity between
wbpM
O11
and
wbpM
O5
, previously shown by others to be present in all 20
P. aeruginosa
serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of
P. aeruginosa
serogroup O11 O antigen.
Mutagenesis of the
Agrobacterium
VirE2 Single-Stranded DNA-Binding Protein Identifies Regions Required for Self-Association and Interaction with VirE1 and a Permissive Site for Hybrid Protein Construction
Authors: Zhou Xue-Rong , Christie Peter J. 
Published: December 2019

ABSTRACT

The VirE2 single-stranded DNA-binding protein (SSB) of
Agrobacterium tumefaciens
is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast
Saccharomyces cerevisiae
correlated with the abundance of the mutant proteins in
A. tumefaciens
, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A

P
virB

::
virE2
construct yielded functional VirE2 protein as defined by complementation of a
virE2
null mutation. By contrast,

P
virE

or

P
lac

promoter constructs yielded functional VirE2 only if
virE1
was coexpressed with
virE2
. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in
A. tumefaciens
, and (iv) restore wild-type virulence to a
virE2
null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.
An Additional Regulatory Gene for Actinorhodin Production inStreptomyces lividansInvolves a LysR-Type Transcriptional Regulator
Authors: Martínez-Costa Oscar H. , Martín-Triana Angel J. , Martínez Eduardo , Fernández-Moreno Miguel A. , Malpartida Francisco 
Published: December 2019

ABSTRACTThe sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream ofactVB-orf6fromStreptomyces coelicolorA3(2) reveals six complete open reading frames, namedorf7toorf12. The deduced amino acid sequences fromorf7,orf10, andorf11show significant similarities with the following products in the databases: a putative protein from theS. coelicolorSCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively. The deduced product oforf8reveals low similarities with several methyltransferases from different sources, whileorf9andorf12products show no similarities with other known proteins. Disruptions oforf10andorf11genes inS. coelicolorappear to have no significant effect on the production of actinorhodin. Nevertheless, disruption or deletion oforf10inStreptomyces lividanscauses actinorhodin overproduction. The introduction of extra copies oforf10andorf11genes in anS. coelicolor actIIImutant restores the ability to produce actinorhodin. Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulatesorf11transcription, expression of which might require the presence of an unknown inducer. No DNA target for Orf10 protein was found within theactcluster.
Genetic Characterization of a Cell Envelope-Associated Proteinase from
Lactobacillus helveticus
CNRZ32
Authors: Pederson Jeffrey A. , Mileski Gerald J. , Weimer Bart C. , Steele James L. 
Published: December 2019

ABSTRACT

A cell envelope-associated proteinase gene (
prtH
) was identified in
Lactobacillus helveticus
CNRZ32. The
prtH
gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the
prtH
product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that
prtH
is not broadly distributed within
L. helveticus
. A
prtH
deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of α
s1
-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its
prtH
deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.
Gene Organization of thednaARegion ofWolbachia
Authors: Sun Ling V. , Babaratsas Alekos , Savakis Charalambos , O’Neill Scott L. , Bourtzis Kostas 
Published: December 2019

ABSTRACTThednaAregion ofWolbachia, an intracellular bacterial parasite of insects, is unique. AglnAcognate was found upstream of thednaAgene, while neither of the two open reading frames detected downstream ofdnaAhas any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.
The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto Are Secreted in Culture from Pseudomonas syringaePathovars via the Hrp (Type III) Protein Secretion System in a Temperature- and pH-Sensitive Manner
Authors: van Dijk Karin , Fouts Derrick E. , Rehm Amos H. , Hill Angela R. , Collmer Alan , Alfano James R. 
Published: December 2019

ABSTRACT
We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways fromPseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22°C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel theP. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name ofhrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.
Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It
Authors: Chebrou Hervé , Hurtubise Yves , Barriault Diane , Sylvestre Michel 
Published: December 2019

ABSTRACTIn this work, we have purified the His-tagged oxygenase (ht-oxygenase) component ofRhodococcus globerulusP6 biphenyl dioxygenase. The α or β subunit of P6 oxygenase was exchanged with the corresponding subunit ofPseudomonassp. strain LB400 or ofComamonas testosteroniB-356 to create new chimeras that were purified ht-proteins and designated ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356were not expressed active in recombinantEscherichia colicells carrying P6bphA1andbphA2, P6bphA1and LB400bphE, or P6bphA1and B-356bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6and ht-αB-356βP6were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained fromPseudomonas putidaKT2440 carrying constructions derived from the new shuttleE. coli-Pseudomonasvector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.
Catalytic Properties of the 3-Chlorocatechol-Oxidizing 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Sphingomonas sp. Strain BN6
Authors: Riegert Ulrich , Heiss Gesche , Kuhm Andrea Elisabeth , Müller Claudia , Contzen Matthias , Knackmuss Hans-Joachim , Stolz Andreas 
Published: December 2019

ABSTRACTThe 2,3-dihydroxybiphenyl dioxygenase fromSphingomonassp. strain BN6 (BphC1-BN6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of different substrates and the effects of various inhibitors on BphC1-BN6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid. Cell extracts containing C23O-mt2 or BphC2-BN6 converted the relevant substrates with an almost constant rate for at least 10 min, whereas BphC1-BN6 was inactivated significantly within the first minutes during the turnover of all substrates tested. Furthermore, BphC1-BN6 was much more sensitive than the other two enzymes to inactivation by the Fe(II) ion-chelating compoundo-phenanthroline. The reason for inactivation of BphC1-BN6 appeared to be the loss of the weakly bound ferrous ion, which is the cofactor in the catalytic center. A mutant enzyme of BphC1-BN6 constructed by site-directed mutagenesis showed a higher stability to inactivation byo-phenanthroline and an increased catalytic efficiency for the conversion of 2,3-dihydroxybiphenyl and 3-methylcatechol but was still inactivated during substrate oxidation.
The bvr Locus of Listeria monocytogenes Mediates Virulence Gene Repression by β-Glucosides
Authors: Brehm Klaus , Ripio María-Teresa , Kreft Jürgen , Vázquez-Boland José-Antonio 
Published: December 2019

ABSTRACTThe β-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence geneshlyandplcAinListeria monocytogenesNCTC 7973. This led to the hypothesis that β-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes ifL. monocytogenesis living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressedhly. This suggested that the downregulation of virulence genes by β-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates ofL. monocytogenesby using the PrfA-dependent phospholipase C geneplcBas a reporter. Utilization of any fermentable sugar causedplcBrepression in wild-typeL. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973,plcBwas only repressed by β-glucosides. Thus, the regulation ofL. monocytogenesvirulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to β-glucosides. We have identified inL. monocytogenesa 4-kb operon,bvrABC, encoding an antiterminator of the BglG family (bvrA), a β-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from otherListeriaspp. Transcription ofbvrBwas induced by cellobiose and salicin but not by arbutin. Disruption of thebvroperon by replacing part ofbvrABwith an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that thebvrlocus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found inL. monocytogenesthat is involved in environmental regulation of virulence genes.
Essential Components of the Ti Plasmidtrb System, a Type IV Macromolecular Transporter
Authors: Li Pei-Li , Hwang Ingyu , Miyagi Heather , True Heather , Farrand Stephen K. 
Published: December 2019

ABSTRACT
The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production ofAgrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58ΔaccR mutations intrbB, -C, -D, -E, -L, -F, -G, and -Habolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbImutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation intraI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of thetrb genes except traI and trbJ can be complemented by functions coded for by pAtC58.
Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria
Authors: Hu Ping , Leighton Terrance , Ishkhanova Galina , Kustu Sydney 
Published: December 2019

ABSTRACTPrevious studies showed thatSalmonella typhimuriumapparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation inBacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size ofB. subtilisclearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious inB. subtilis. To rule out the possibility that differences were due to the fact thatB. subtilishas only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase inSalmonella. Studies of theS. typhimurium gdhstrain in ammonia-limited continuous culture and ofgdh glnAdouble-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results withB. subtilisare that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.
Functional and Transcriptional Analyses of a Fengycin Synthetase Gene, fenC, from Bacillus subtilis
Authors: Lin Tsuey-Pin , Chen Chyi-Liang , Chang Li-Kwan , Tschen Johannes Scheng-Ming , Liu Shih-Tung 
Published: December 2019

ABSTRACTA 37-kb DNA fragment containing five fengycin synthetase genes, includingfenC,fenD,fenE,fenA, andfenB, was cloned and sequenced. Among these genes,fenCencodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activatel-glutamic acid andl-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon offenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon offenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription offenCis positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis byB. subtilisF29-3.
In Vitro Analysis of the Butyrolactone Autoregulator Receptor Protein (FarA) of Streptomyces lavendulae FRI-5 Reveals that FarA Acts as a DNA-Binding Transcriptional Regulator That Controls Its Own Synthesis
Authors: Kitani Shigeru , Kinoshita Hiroshi , Nihira Takuya , Yamada Yasuhiro 
Published: December 2019

ABSTRACT
FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farAtranscripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farAtranslation start codon and 4 bp downstream from an Escherichia coli ς70-like −10 recognition region. The FarA-binding sequence overlaps this −10 region and contains thefarA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.