bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of
Sphingomonas paucimobilis
SYK-6
Authors: Masai Eiji , Shinohara Shouji , Hara Hirofumi , Nishikawa Seiji , Katayama Yoshihiro , Fukuda Masao 
Published: December 2019

ABSTRACT

Sphingomonas paucimobilis
SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb
Eco
RI fragment carrying the protocatechuate 4,5-dioxygenase gene (
ligAB
) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the
ligI
gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The
ligI
gene is thought to be transcribed divergently from
ligAB
and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The
ligI
gene product (LigI), expressed in
Escherichia coli
, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the

K
m

values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of
Pseudomonas ochraceae
(K. Maruyama, J. Biochem. 93:557–565, 1983) and
Comamonas testosteroni
(P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the
ligI
gene in
S. paucimobilis
SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the
ligI
gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.
Construction and Analysis of a
Streptococcus parasanguis recA
Mutant: Homologous Recombination Is Not Required for Adhesion in an In Vitro Tooth Surface Model
Authors: Froeliger Eunice H. , Tomich Mladen , Fives-Taylor Paula 
Published: December 2019

ABSTRACT

PCR was used to amplify an internal region of the
recA
gene from
Streptococcus parasanguis
FW213. The PCR fragment was used as a probe to recover the entire streptococcal
recA
gene from an
S. parasanguis
genomic library, and the sequence of the gene was determined. The deduced product of the
S. parasanguis recA
gene showed a high degree of amino acid identity with other prokaryotic RecA proteins. The cloned
recA
sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into the
S. parasanguis
chromosome by homologous recombination. Results of Southern hybridizations confirmed the replacement of the wild-type
recA
gene with the mutated allele. The
recA
mutant strain was considerably more sensitive to UV light than the parental strain, and this phenotype was consistent with a mutation in
recA
. The
S. parasanguis recA
mutant showed no reduction in its ability to adhere in the in vitro tooth surface model, saliva-coated hydroxylapatite (SHA), or in its ability to express the fimbria-associated adhesin Fap1. These results demonstrate that in vitro attachment of
S. parasanguis
FW213 to SHA and expression of Fap1 are
recA
independent.
Rickettsia prowazekii
Transports UMP and GMP, but Not CMP, as Building Blocks for RNA Synthesis
Authors: Winkler Herbert H. , Daugherty Robin , Hu Fuquan 
Published: December 2019

ABSTRACT

Rickettsia prowazekii
, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified
R. prowazekii
organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.
Isolation and Characterization of the
cis-trans
-Unsaturated Fatty Acid Isomerase of
Pseudomonas oleovorans
GPo12
Authors: Pedrotta Valerian , Witholt Bernard 
Published: December 2019

ABSTRACT

Pseudomonas oleovorans
contains an isomerase which catalyzes the
cis-trans
conversion of the abundant unsaturated membrane fatty acids 9-
cis
-hexadecenoic acid (palmitoleic acid) and 11-
cis
-octadecenoic acid (vaccenic acid). We purified the isomerase from the periplasmic fraction of
Pseudomonas oleovorans
. The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme. N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme. The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids. In contrast to the in vivo
cis-trans
conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents. Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase. However, when crude membranes from
Pseudomonas
or
Escherichia coli
cells were used as phospholipid sources, a
cis-trans
isomerization was detectable which occurred only in the presence of organic solvents. These results indicate that isolated membranes from
Pseudomonas
or
E. coli
cells must contain factors which, activated by the addition of organic solvents, enable and control the
cis-trans
conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase.
Metabolic Imbalance and Sporulation in an Isocitrate Dehydrogenase Mutant of
Bacillus subtilis
Authors: Matsuno Kiyoshi , Blais Tessa , Serio Alisa W. , Conway Tyrrell , Henkin Tina M. , Sonenshein Abraham L. 
Published: December 2019

ABSTRACT

A
Bacillus subtilis
mutant with a deletion in the
citC
gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (
citZ
), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the
sof-1
mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a
citC
mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.
Complex Evolutionary Patterns of tRNA
UAA
Leu
Group I Introns in Cyanobacterial Radiation
Authors: Rudi Knut , Jakobsen Kjetill S. 
Published: December 2019

ABSTRACT

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNA
UAA
Leu
genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNA
UAA
Leu
genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNA
UAA
Leu
group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNA
UAA
Leu
genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNA
UAA
Leu
group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.
Genetic Analysis of a Chromosomal Region Containing
vanA
and
vanB
, Genes Required for Conversion of Either Ferulate or Vanillate to Protocatechuate in
Acinetobacter
Authors: Segura Ana , Bünz Patricia V. , D’Argenio David A. , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

VanA and VanB form an oxygenative demethylase that converts vanillate to protocatechuate in microorganisms. Ferulate, an abundant phytochemical, had been shown to be metabolized through a vanillate intermediate in several
Pseudomonas
isolates, and biochemical evidence had indicated that vanillate also is an intermediate in ferulate catabolism by
Acinetobacter
. Genetic evidence supporting this conclusion was obtained by characterization of mutant
Acinetobacter
strains blocked in catabolism of both ferulate and vanillate. Cloned
Acinetobacter vanA
and
vanB
were shown to be members of a chromosomal segment remote from a supraoperonic cluster containing other genes required for completion of the catabolism of ferulate and its structural analogs, caffeate and coumarate, through protocatechuate. The nucleotide sequence of DNA containing
vanA
and
vanB
demonstrated the presence of genes that, on the basis of nucleotide sequence similarity, appeared to be associated with transport of aromatic compounds, metabolism of such compounds, or iron scavenging. Spontaneous deletion of 100 kb of DNA containing this segment does not impede the growth of cells with simple carbon sources other than vanillate or ferulate. Additional spontaneous mutations blocking
vanA
and
vanB
expression were shown to be mediated by IS
1236
, including insertion of the newly discovered composite transposon Tn
5613
. On the whole,
vanA
and
vanB
appear to be located within a nonessential genetic region that exhibits considerable genetic malleability in
Acinetobacter
. The overall organization of genes neighboring
Acinetobacter vanA
and
vanB
, including a putative transcriptional regulatory gene that is convergently transcribed and overlaps
vanB
, is conserved in
Pseudomonas aeruginosa
but has undergone radical rearrangement in other
Pseudomonas
species.
cpmA
, a Gene Involved in an Output Pathway of the Cyanobacterial Circadian System
Authors: Katayama Mitsunori , Tsinoremas Nicholas F. , Kondo Takao , Golden Susan S. 
Published: December 2019

ABSTRACT

We generated random mutations in
Synechococcus
sp. strain PCC 7942 to look for genes of output pathways in the cyanobacterial circadian system. A derivative of transposon Tn
5
was introduced into the chromosomes of reporter strains in which cyanobacterial promoters drive the
Vibrio harveyi luxAB
genes and produce an oscillation of bioluminescence as a function of circadian gene expression. Among low-amplitude mutants, one mutant, tnp6, had an insertion in a 780-bp open reading frame. The tnp6 mutation produced an altered circadian phasing phenotype in the expression rhythms of
psbAI
::
luxAB
,
psbAII
::
luxAB
, and
kaiA
::
luxAB
but had no or little effect on those of
psbAIII
::
luxAB
,
purF
::
luxAB
,
kaiB
::
luxAB
,
rpoD2
::
luxAB
,
ndhD
::
luxAB
, and
conII
::
luxAB
. This suggests that the interrupted gene in tnp6, named
cpmA
(circadian phase modifier), is part of a circadian output pathway that regulates the expression rhythms of
psbAI
,
psbAII
, and
kaiA
.
Comparison of the Heme Iron Utilization Systems of Pathogenic Vibrios
Authors: O’Malley S. M. , Mouton S. L. , Occhino D. A. , Deanda M. T. , Rashidi J. R. , Fuson K. L. , Rashidi C. E. , Mora M. Y. , Payne S. M. , Henderson D. P. 
Published: December 2019

ABSTRACT

Vibrio alginolyticus
,
Vibrio fluvialis
, and
Vibrio parahaemolyticus
utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several
Vibrio cholerae
heme iron utilization genes. A
V. parahaemolyticus
gene that performed the function of
V. cholerae hutA
was isolated. A portion of the
tonB1
locus of
V. parahaemolyticus
was sequenced and found to encode proteins similar in amino acid sequence to
V. cholerae
HutW, TonB1, and ExbB1. A recombinant plasmid containing the
V. cholerae tonB1
and
exbB1D1
genes complemented a
V. alginolyticus
heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly
V. parahaemolyticus
and
V. alginolyticus
, are similar at the DNA level, the functional level, and, in the case of
V. parahaemolyticus
, the amino acid sequence or protein level to that of
V. cholerae.
Role in Cell Permeability of an Essential Two-Component System inStaphylococcus aureus
Authors: Martin Patrick K. , Li Tong , Sun DongXu , Biek Donald P. , Schmid Molly B. 
Published: December 2019

ABSTRACTA temperature-sensitive lethal mutant ofStaphylococcus aureuswas found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded byyycFG; orthologues ofyycFGcould be identified in the genomes ofBacillus subtilisand other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either theyycFor theyycGcoding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of theermBresistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.
Role for the
oxyS
Gene in Regulation of Intracellular Hydrogen Peroxide in
Escherichia coli
Authors: González-Flecha Beatriz , Demple Bruce 
Published: December 2019

ABSTRACT

Intracellular hydrogen peroxide is regulated in
Escherichia coli
by OxyR in response to the metabolic production of H
2
O
2
. Here, we show that the untranslated
oxyS
RNA controlled by OxyR has a role in this regulation. The
oxyS
transcript appears to affect the metabolic output of H
2
O
2
rather than the removal of H
2
O
2
by catalases-hydroperoxidases.
Activation of
Escherichia coli leuV
Transcription by FIS
Authors: Ross Wilma , Salomon Julia , Holmes Walter M. , Gourse Richard L. 
Published: December 2019

ABSTRACT

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNA
Leu
species in
Escherichia coli
, tRNA
1
Leu
. However, no evidence for direct involvement of FIS in tRNA
1
Leu
expression has been reported. We show here that FIS binds to a site upstream of the
leuV
promoter (centered at −71) and that it directly stimulates
leuV
transcription in vitro. A mutation in the FIS binding site reduces transcription from a
leuV
promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to
leuV
expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the
leuV
promoter, having a downstream protection boundary of ∼−7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of
leuV
transcription.
Characterization of the Major Superoxide Dismutase of
Staphylococcus aureus
and Its Role in Starvation Survival, Stress Resistance, and Pathogenicity
Authors: Clements Mark O. , Watson Sean P. , Foster Simon J. 
Published: December 2019

ABSTRACT

A
Staphylococcus aureus
mutant (SPW1) which is unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to the
sodA
family of manganese-dependent superoxide dismutases (SOD). Whole-cell lysates of the parental 8325-4 strain demonstrated three zones of SOD activity by nondenaturing gel electrophoresis. The activities of two of these zones were dependent on manganese for activity and were absent in SPW1. The levels of SOD activity and
sodA
expression were growth-phase dependent, occurring most during postexponential phase. This response was also dependent on the level of aeration of the culture, with highest activity and expression occurring only under high aeration. Expression of
sodA
and, consequently, SOD activity could be induced by methyl viologen but only during the transition from exponential- to postexponential-phase growth. SPW1 was less able to survive amino acid limitation and acid stress but showed no alteration in pathogenicity in a mouse abscess model of infection compared to the parental strain 8325-4.
Regulation of
podJ
Expression during the
Caulobacter crescentus
Cell Cycle
Authors: Crymes William B. , Zhang Daolong , Ely Bert 
Published: December 2019

ABSTRACT

The polar organelle development gene,
podJ
, is expressed during the swarmer-to-stalked cell transition of the
Caulobacter crescentus
cell cycle. Mutants with insertions that inactivate the
podJ
gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of
podJ
results in a lethal cell division defect. Nucleotide sequence analysis of the
podJ
promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the
podJ
promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of
podJ
promoter activity is dependent on the CtrA binding site, the temporal control of
podJ
promoter expression is dependent on other factors.
Structural and Functional Analyses of Photosynthetic Regulatory Genes
regA
and
regB
from
Rhodovulum sulfidophilum
,
Roseobacter denitrificans
, and
Rhodobacter capsulatus
Authors: Masuda Shinji , Matsumoto Yumi , Nagashima Kenji V. P. , Shimada Keizo , Inoue Kazuhito , Bauer Carl E. , Matsuura Katsumi 
Published: December 2019

ABSTRACT

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria
Rhodovulum sulfidophilum
and
Roseobacter denitrificans
, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from
Rhodobacter capsulatus
and
Rhodobacter sphaeroides
. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A
Rhodovulum sulfidophilum
mutant lacking
regA
or
regB
was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in
Rhodobacter
species.
Rhodobacter capsulatus regA
- or
regB
-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with
reg
genes from
Rhodovulum sulfidophilum
and
Roseobacter denitrificans
. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.