bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Function of the
pdxY
Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in
Escherichia coli
K-12
Authors: Yang Yong , Tsui Ho-Ching Tiffany , Man Tsz-Kwong , Winkler Malcolm E.
Published: December 2019

pdxK
encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that
pdxK
null mutants still contain PL kinase activity led to the hypothesis that
Escherichia coli
K-12 contains at least one other B
6
-vitamer kinase. Here we support this hypothesis by identifying the
pdxY
gene (formally, open reading frame
f287b
) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete
E. coli
genome. Minimal clones of
pdxY
+
overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into
pdxY
and crossed the resulting
pdxY
::ΩKan
r
mutation into the bacterial chromosome of a
pdxB
mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of
pdxK
and
pdxY
single and double mutants. Significantly, the requirement of the
pdxB pdxK pdxY
triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B
6
vitamer kinases in
E. coli
and that their function is confined to the PLP salvage pathway. Last, we show that
pdxY
is located downstream from
pdxH
(encoding PNP/PMP oxidase) and essential
tyrS
(encoding aminoacyl-tRNA
Tyr
synthetase) in a multifunctional operon.
pdxY
is completely cotranscribed with
tyrS
, but about 92% of
tyrS
transcripts terminate at a putative Rho-factor-dependent attenuator located in the
tyrS-pdxY
intercistronic region.
Efficiency of T4 Gene
60
Translational Bypassing
Authors: Maldonado Rafael , Herr Alan J.
Published: December 2019

Ribosomes translating bacteriophage T4 gene
60
mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene
60
mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene
60
(which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of ∼50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.
Thioredoxin Is an Essential Protein Induced by Multiple Stresses in
Bacillus subtilis
Authors: Scharf Christian , Riethdorf Sabine , Ernst Henrik , Engelmann Susanne , Völker Uwe , Hecker Michael
Published: December 2019

Thioredoxin, a small, ubiquitous protein which participates in redox reactions through the reversible oxidation of its active center dithiol to a disulfide, is an essential protein in
Bacillus subtilis
. A variety of stresses, including heat or salt stress or ethanol treatment, strongly enhanced the synthesis of thioredoxin in
B. subtilis
. The stress induction of the monocistronic
trxA
gene encoding thioredoxin occurs at two promoters. The general stress sigma factor, ς
B
, was required for the initiation of transcription at the upstream site, S
B
, and the promoter preceding the downstream start site, S
A
, was presumably recognized by the vegetative sigma factor, ς
A
. In contrast to the heat-inducible, ς
A
-dependent promoters preceding the chaperone-encoding operons
groESL
and
dnaK
, no CIRCE (for controlling inverted repeat of chaperone expression) was present in the vicinity of the start site, S
A
. The induction patterns of the promoters differed, with the upstream promoter displaying the typical stress induction of ς
B
-dependent promoters. Transcription initiating at S
A
, but not at S
B
, was also induced after treatment with hydrogen peroxide or puromycin. Such a double control of stress induction at two different promoters seems to be typical of a subgroup of class III heat shock genes of
B. subtilis
, like
clpC
, and it either allows the cells to raise the level of the antioxidant thioredoxin after oxidative stress or allows stressed cells to accumulate thioredoxin. These increased levels of thioredoxin might help stressed
B. subtilis
cells to maintain the native and reduced state of cellular proteins.
Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of
Saccharomyces cerevisiae
Authors: Nishimura Ken , Igarashi Kazuei , Kakinuma Yoshimi
Published: December 2019

A vacuolar H
+
-ATPase-negative mutant of
Saccharomyces cerevisiae
was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.
Identification of the Ω4400 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
Authors: Brandner Janine P. , Kroos Lee
Published: December 2019

Ω4400 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
genome that fuses
lacZ
expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn
5 lac
Ω4400. The DNA upstream of the Ω4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn
5 lac
Ω4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by β-galactosidase production in cells containing Tn
5 lac
Ω4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of −101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to −76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the
M. xanthus sigB
or
sigC
gene did not affect Tn
5 lac
Ω4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any
M. xanthus
promoter transcribed by a known form of RNA polymerase. However, the Ω4400 promoter does contain the sequence 5′-CATCCCT-3′ centered at −49 and the C-signal-dependent Ω4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the −10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the
cis
-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.
Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport ofMethanosarcina mazeiGö1
Authors: Abken Hans-Jörg , Tietze Mario , Brodersen Jens , Bäumer Sebastian , Beifuss Uwe , Deppenmeier Uwe
Published: December 2019

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes ofMethanosarcina mazeiGö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F420H2dehydrogenase fromM. mazeiGö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F420H2was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. TheKmvalues for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F420-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport ofM. mazeiGö1.
Availability of O
2
as a Substrate in the Cytoplasm of Bacteria under Aerobic and Microaerobic Conditions
Authors: Arras Tanja , Schirawski Jan , Unden Gottfried
Published: December 2019

The growth rates of
Pseudomonas putida
KT2442 and mt-2 on benzoate, 4-hydroxybenzoate, or 4-methylbenzoate showed an exponential decrease with decreasing oxygen tensions (partial O
2
tension [pO
2
] values). The oxygen tensions resulting in half-maximal growth rates were in the range of 7 to 8 mbar of O
2
(corresponding to 7 to 8 μM O
2
) (1 bar = 10
5
Pa) for aromatic compounds, compared to 1 to 2 mbar for nonaromatic compounds like glucose or succinate. The decrease in the growth rates coincided with excretion of catechol or protocatechuate, suggesting that the activity of the corresponding oxygenases became limiting. The experiments directly establish that under aerobic and microaerobic conditions (about 10 mbar of O
2
), the diffusion of O
2
into the cytoplasm occurs at high rates sufficient for catabolic processes. This is in agreement with calculated O
2
diffusion rates. Below 10 mbar of O
2
, oxygen became limiting for the oxygenases, probably due to their high

K
m

values, but the diffusion of O
2
into the cytoplasm presumably should be sufficiently rapid to maintain ambient oxygen concentrations at oxygen tensions as low as 1 mbar of O
2
. The consequences of this finding for the availability of O
2
as a substrate or as a regulatory signal in the cytoplasm of bacterial cells are discussed.
Interaction of Native and Mutant MecI Repressors with Sequences That Regulate
mecA
, the Gene Encoding Penicillin Binding Protein 2a in Methicillin-Resistant Staphylococci
Authors: Sharma Vijay K. , Hackbarth Corinne J. , Dickinson Tanja M. , Archer Gordon L.
Published: December 2019

Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for β-lactam antibiotics. The gene encoding PBP2a,
mecA
, is transcriptionally regulated in some clinical isolates by
mecR1
and
mecI
, genes divergently transcribed from
mecA
that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediated
mecA
transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted
mecA
−10 and the
mecR1
−35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire
mecA-mecR1
intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed
mecA
transcription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.
Developmental Regulation of Transcription of
whiE
, a Locus Specifying the Polyketide Spore Pigment in
Streptomyces coelicolor
A3(2)
Authors: Kelemen Gabriella H. , Brian Paul , Flärdh Klas , Chamberlin Leony , Chater Keith F. , Buttner Mark J.
Published: December 2019

whiE
is a complex locus that specifies the polyketide spore pigment in
Streptomyces coelicolor
A3(2). Two divergently oriented promoters,
whiEP1
and
whiEP2
, were identified in the
whiE
gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated;
whiEP1
and
whiEP2
transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known “early”
whi
genes required for sporulation septum formation (
whiA
, -
B
, -
G
, -
H
, -
I
, and -
J
). Mutation of the late sporulation-specific sigma factor gene,
sigF
, had no effect on the activity of
whiEP1
but blocked transcription from
whiEP2
. However, ς
F
-containing holoenzyme was not sufficient to direct transcription of
whiEP2
in vitro. The
whiEP2
promoter controls expression of
whiE
ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of
whiE
ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779–2791, 1995). Consistent with these observations, construction of a
sigF
null mutant of
S. coelicolor
M145 caused the same change in spore color, showing that disruption of
sigF
in
S. coelicolor
changes the nature of the spore pigment rather than preventing its synthesis altogether.
Kinetics of
nirS
Expression (Cytochrome
cd
1
Nitrite Reductase) in
Pseudomonas stutzeri
during the Transition from Aerobic Respiration to Denitrification: Evidence for a Denitrification-Specific Nitrate- and Nitrite-Responsive Regulatory System
Authors: Härtig Elisabeth , Zumft Walter G.
Published: December 2019

After shifting an oxygen-respiring culture of
Pseudomonas stutzeri
to nitrate or nitrite respiration, we directly monitored the expression of the
nirS
gene by mRNA analysis.
nirS
encodes the 62-kDa subunit of the homodimeric cytochrome
cd
1
nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that
nirS
, together with
nirT
and
nirB
(which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of
nirS
lasting for about 30 min. The continued transcription of the
nirS
operon required the presence of nitrate or nitrite. This anaerobically manifested
N
-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the
norCB
operon, encoding the NO reductase complex, and the
nosZ
gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.
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

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

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

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

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

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

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

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

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

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

A 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

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

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

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

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

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.
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

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

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

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

The 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

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.