bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of the Pyruvate Dehydrogenase Multienzyme Complex of
Zymomonas mobilis
and Identification and Sequence Analysis of the Corresponding Genes
Authors: Neveling Ute , Klasen Ralf , Bringer-Meyer Stephanie , Sahm Hermann 
Published: December 2019

ABSTRACT

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium
Zymomonas mobilis
was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of
Z. mobilis
. The genes
pdhA
α (1,065 bp) and
pdhA
β (1,389 bp), encoding the E1α and E1β subunits of the E1 component, were located downstream of the gene encoding enolase. The
pdhB
(1,323 bp) and
lpd
(1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order
pdhB
-ORF2-
lpd
. Highest similarities of the gene products of the
pdhA
α,
pdhA
β, and
pdhB
genes were found with the corresponding enzymes of
Saccharomyces cerevisiae
and other eukaryotes. Like the dihydrolipoamide acetyltransferases of
S. cerevisiae
and numerous other organisms, the product of the
pdhB
gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.
The
bldD
Gene of
Streptomyces coelicolor
A3(2): a Regulatory Gene Involved in Morphogenesis and Antibiotic Production
Authors: Elliot Marie , Damji Farzana , Passantino Rosa , Chater Keith , Leskiw Brenda 
Published: December 2019

ABSTRACT

The
bld
mutants of
Streptomyces coelicolor
A3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type
S. coelicolor
DNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known
bldD
mutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated
bldD
, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the
bldD
-containing fragment amplified from the chromosome of a
bldD
mutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of
bldD
transcript levels in the
bldD
+
and
bldD
mutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of
bldD
transcripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the
bldD
start codon and 7 nucleotides downstream from −10 and −35 sequences resembling
E. coli
-like streptomycete promoters.
The
bldB
Gene Encodes a Small Protein Required for Morphogenesis, Antibiotic Production, and Catabolite Control in
Streptomyces coelicolor
Authors: Pope Margaret K. , Green Brian , Westpheling Janet 
Published: December 2019

ABSTRACT

Mutants blocked at the earliest stage of morphological development in
Streptomyces
species are called
bld
mutants. These mutants are pleiotropically defective in the initiation of development, the ability to produce antibiotics, the ability to regulate carbon utilization, and the ability to send and/or respond to extracellular signals. Here we report the identification and partial characterization of a 99-amino-acid open reading frame (ORF99) that is capable of restoring morphogenesis, antibiotic production, and catabolite control to all of the
bldB
mutants. Of the existing
bld
mutants,
bldB
is of special interest because the phenotype of this mutant is the most pleiotropic. DNA sequence analysis of ORF99 from each of the existing
bldB
mutants identified base changes either within the coding region of the predicted protein or in the regulatory region of the gene. Primer extension analysis identified an apparent transcription start site. A promoter fusion to the
xylE
reporter gene showed that expression of
bldB
is apparently temporally regulated and that the
bldB
gene product is involved in the regulation of its own expression.
Melanin Biosynthesis in
Cryptococcus neoformans
Authors: Williamson Peter R. , Wakamatsu Kazumasa , Ito Shosuke 
Published: December 2019

ABSTRACT

Pigment production by
Cryptococcus neoformans
is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.
A Surface-Exposed Region of a Novel Outer Membrane Protein (P66) ofBorreliaspp. Is Variable in Size and Sequence
Authors: Bunikis Jonas , Luke Catherine J. , Bunikiene Elena , Bergström Sven , Barbour Alan G. 
Published: December 2019

ABSTRACTA model of the 66-kDa outer membrane protein (P66) of Lyme diseaseBorreliaspp. predicts a surface-exposed loop near the C terminus. This region contains an antigen commonly recognized by sera from Lyme disease patients. In the present study, this region of P66 and homologous proteins of otherBorreliaspp. were further investigated by using monoclonal antibodies, epitope mapping of P66 ofBorrelia burgdorferi, and DNA sequencing. A monoclonal antibody specific forB. burgdorferibound to the portion of P66 that was accessible to proteolysis in situ. The linear epitope for the antibody was mapped within a variable segment of the surface-exposed region. To further study this protein, the complete gene ofBorrelia hermsiifor a protein homologous to P66 was cloned. The deduced protein was 589 amino acids in length and 58% identical to P66 ofB. burgdorferi. TheB. hermsiiP66 protein was predicted to have a surface-exposed region in the same location as that ofB. burgdorferi’s P66 protein. With primers designed on the basis of conserved sequences and PCR, we identified and cloned the same regions of P66 proteins ofBorrelia turicatae,Borrelia parkeri,Borrelia coriaceae, andBorrelia anserina. The deduced protein sequences from all species demonstrated two conserved hydrophobic regions flanking a surface-exposed loop. The loop sequences were highly variable between differentBorreliaspp. in both sequence and size, varying between 35 and 45 amino acids. Although the actual function of P66 ofBorreliaspp. is unknown, the results suggest that its surface-exposed region is subject to selective pressure.
The CIRCE Element and Its Putative Repressor Control Cell Cycle Expression of the
Caulobacter crescentus groESL
Operon
Authors: Baldini Regina Lúcia , Avedissian Marcelo , Gomes Suely Lopes 
Published: December 2019

ABSTRACT

The
groESL
operon is under complex regulation in
Caulobacter crescentus
. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the
groE
genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of
groESL
contains both a ς
32
-like promoter and a CIRCE element. Overexpression of
C. crescentus
ς
32
gives rise to higher levels of GroEL and increased levels of the
groESL
transcript coming from the ς
32
-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for this
cis
-acting element in the expression of
groESL
only at normal growth temperatures, with a minor effect on heat shock induction. Furthermore,
groESL-lacZ
transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an
hrcA
null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout the
Caulobacter
cell cycle. These results indicate a negative role for the
hrcA
gene product and the CIRCE element in the temporal control of the
groESL
operon.
Forespore Expression and Processing of the SigE Transcription Factor in Wild-Type and Mutant
Bacillus subtilis
Authors: Ju Jingliang , Luo Tingqiu , Haldenwang W. G. 
Published: December 2019

ABSTRACT

ς
E
is a mother cell-specific transcription factor of sporulating
Bacillus subtilis
that is derived from an inactive precursor protein (pro-ς
E
). To examine the process that prevents ς
E
activity from developing in the forespore, we fused the ς
E
structural gene (
sigE
) to forespore-specific promoters (

P
dacF

and

P
spoIIIG

), placed these fusions at sites on the
B. subtilis
chromosome which translocate into the forespore either early or late, and used Western blot analysis to monitor SigE accumulation and pro-ς
E
processing.
sigE
alleles, placed at sites which entered the forespore early, were found to generate more protein product than the same fusion placed at a late entering site. SigE accumulation and processing in the forespore were enhanced by null mutations in
spoIIIE
, a gene whose product is essential for translocation of the distal portion of the
B. subtilis
chromosome into the forespore. In other experiments, a chimera of pro-ς
E
and green fluorescence protein, previously shown to be unprocessed if it is synthesized within the forespore, was found to be processed in this compartment if coexpressed with the gene for the pro-ς
E
-processing enzyme, SpoIIGA. The need for
spoIIGA
coexpression is obviated in the absence of SpoIIIE. We interpret these results as evidence that selective degradation of both SigE and SpoIIGA prevent mature ς
E
from accumulating in the forespore compartment of wild-type
B. subtilis
. Presumably, a gene(s) located at a site that is distal to the origin of chromosome transfer is responsible for this phenomenon when it is translocated and expressed in the forespore.
The
Rhizobium etli rpoN
Locus: DNA Sequence Analysis and Phenotypical Characterization of
rpoN
,
ptsN
, and
ptsA
Mutants
Authors: Michiels Jan , Van Soom Tom , D’hooghe Inge , Dombrecht Bruno , Benhassine Traki , de Wilde Petra , Vanderleyden Jos 
Published: December 2019

ABSTRACT

The
rpoN
region of
Rhizobium etli
was isolated by using the
Bradyrhizobium japonicum rpoN1
gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258,
rpoN
, ORF191, and
ptsN
, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and
ptsN
are homologous to conserved ORFs found downstream from
rpoN
genes in other bacterial species. Unlike in most other microorganisms,
rpoN
and ORF191 are separated by approximately 1.6 kb. The
R. etli rpoN
gene was shown to control in free-living conditions the production of melanin, the activation of
nifH
, and the metabolism of C
4
-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the
rpoN
gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the
ptsN
gene resulted in a decrease of melanin synthesis and
nifH
expression. In a search for additional genes controlling the synthesis of melanin, an
R. etli
mutant carrying a Tn
5
insertion in
ptsA
, a gene homologous to the
Escherichia coli
gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The
R. etli ptsA
mutant also displayed reduced expression of
nifH
. The
ptsN
and
ptsA
mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C
4
-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that
ptsN
and
ptsA
are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.
Functions Encoded by Pyrrolnitrin Biosynthetic Genes from
Pseudomonas fluorescens
Authors: Kirner Sabine , Hammer Philip E. , Hill D. Steven , Altmann Annett , Fischer Ilona , Weislo Laura J. , Lanahan Mike , van Pée Karl-Heinz , Ligon James M. 
Published: December 2019

ABSTRACT

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes,
prnABCD
, from
Pseudomonas fluorescens
that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each
prn
gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type
Pseudomonas fluorescens
, but absent from a mutant from which the entire
prn
gene region had been deleted. The
prnA
gene product catalyzes the chlorination of
l
-tryptophan to form 7-chloro-
l
-tryptophan. The
prnB
gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-
l
-tryptophan to monodechloroaminopyrrolnitrin. The
prnC
gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The
prnD
gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the
prn
genes in the operon is identical to the order of the reactions in the biosynthetic pathway.
Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in
Ralstonia eutropha
Authors: Slater Steven , Houmiel Kathryn L. , Tran Minhtien , Mitsky Timothy A. , Taylor Nancy B. , Padgette Stephen R. , Gruys Kenneth J. 
Published: December 2019

ABSTRACT

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism’s intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes.
Ralstonia eutropha
produces both the homopolymer poly-β-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form β-ketovaleryl-CoA. This activity has generally been attributed to the β-ketothiolase encoded by
R. eutropha phbA
. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of
bktB
, which encodes a β-ketothiolase from
R. eutropha
that is capable of forming β-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of β-hydroxyvalerate derived from propionyl-CoA. We also report an additional β-ketothiolase, designated BktC, that probably serves as a secondary route toward β-hydroxyvalerate production.
A Period-Extender Gene,
pex
, That Extends the Period of the Circadian Clock in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Kutsuna Shinsuke , Kondo Takao , Aoki Setsuyuki , Ishiura Masahiro 
Published: December 2019

ABSTRACT

We cloned the pS1K1 plasmid in the process of apparently “complementing” a circadian clock mutant of cyanobacterium
Synechococcus
sp. strain PCC 7942, SP22, which has a 22-h period (T. Kondo, N. F. Tsinoremas, S. S. Golden, C. H. Johnson, S. Kutsuna, and M. Ishiura, Science 266:1233–1236, 1994). Sequence analysis revealed that SP22 did not have a mutation in the genomic DNA segment carried on pS1K1, and the
sp22
mutation was later found in a recently cloned new clock gene,
kaiC
. Therefore, the period-extender gene
pex
that was carried on pS1K1 was a suppressor gene for the
sp22
mutation. The
pex
gene encoded a protein of 148 amino acid residues. No meaningful homologs were found in DNA or protein databases including the
Synechocystis
genome database. The
pex
gene was transcribed from 129 and 164 bp upstream of the translation initiation codon as 0.6-kb transcripts. The Pex protein was detected as a fusion protein with a molecular mass of 15 kDa by the epitope tag fusion method using a c-Myc epitope tag. Disruption of the
pex
gene in wild-type cells shortened the period of the rhythms by 1 h, although it did not affect other properties of the rhythms, whereas its overexpression extended the period by 3 h with a concomitant reduction in the amplitude of the rhythms. In various clock mutants examined, overexpression caused arrhythmicity. Thus, Pex is likely to function as a modifier of the circadian clock in
Synechococcus.
Inorganic Polyphosphate in
Escherichia coli
: the Phosphate Regulon and the Stringent Response
Authors: Rao Narayana N. , Liu Shengjiang , Kornberg Arthur 
Published: December 2019

ABSTRACT

Escherichia coli
transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P
i
), in media deficient in both P
i
and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a
phoB
-containing plasmid,
phoB
mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during P
i
starvation). Hence, accumulation of polyP requires a functional
phoB
gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.
The
Bacillus subtilis galE
Gene Is Essential in the Presence of Glucose and Galactose
Authors: Krispin Oliver , Allmansberger Rudolf 
Published: December 2019

ABSTRACT

Bacillus subtilis
is unable to grow by consuming galactose because it is unable to transport it into the cell. The transcription of
galE
is not influenced by galactose but is repressed by glucose. Galactose is toxic for
galE
-negative bacteria because it results in elevated levels of metabolic intermediates. These negative effects are reduced in
galK
and
galT
mutants. Glucose is also toxic for
galE
-negative strains.
Mutational Analysis of the
Chlamydia trachomatis
rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro
Authors: Tan Ming , Gaal Tamas , Gourse Richard L. , Engel Joanne N. 
Published: December 2019

ABSTRACT

We have characterized the
Chlamydia trachomatis
ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified
C. trachomatis
RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous to
Escherichia coli
promoters recognized by E-ς
70
. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for
C. trachomatis
RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by
C. trachomatis
and
E. coli
RNA polymerases. We propose that this region functions as an UP element.
Cloning and Characterization of a Gene Encoding the Major Surface Protein of the Bacterial EndosymbiontWolbachia pipientis
Authors: Braig Henk R. , Zhou Weiguo , Dobson Stephen L. , O’Neill Scott L. 
Published: December 2019

ABSTRACTThe maternally inherited intracellular symbiontWolbachia pipientisis well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressedWolbachiaproteins from infectedDrosophilaeggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of thewsppromoter region was also successfully tested inEscherichia coli. Comparison of sequences of this gene from different strains ofWolbachiarevealed a high level of variability. This sequence variation correlated with the ability of certainWolbachiastrains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool forWolbachiastrain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction ofWolbachiawith its host.
The
Streptomyces peucetius dpsY
and
dnrX
Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis
Authors: Lomovskaya Natalia , Doi-Katayama Yukiko , Filippini Sylvia , Nastro Cecilia , Fonstein Leonid , Gallo Mark , Colombo Anna Luisa , Hutchinson C. Richard 
Published: December 2019

ABSTRACT

The
Streptomyces peucetius dpsY
and
dnrX
genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of
dpsY
and
dnrX
. Replacement of
dpsY
with a mutant form disrupted by insertion of the
aphII
neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of
dnrX
in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that
dnrX
, analogous to the role of the
S. peucetius dnrH
gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.