bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes inPseudomonassp. Strain 61-3
Authors: Matsusaki Hiromi , Manji Sumihide , Taguchi Kazunori , Kato Mikiya , Fukui Toshiaki , Doi Yoshiharu
Published: December 2019

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phbandpha) ofPseudomonassp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In thephblocus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), β-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed byphbBPs-phbAPs-phbCPs. Upstream fromphbBPswas found thephbRPsgene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. ThephbRPsgene was found to be transcribed in the opposite direction from the three structural genes. Cloning ofphbRPsin a relatively high-copy vector inPseudomonassp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptionalphbpromoter-lacZfusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPsis a positive regulatory protein controlling the transcription ofphbBACPsin this bacterium. In thephalocus, two genes encoding PHA synthases (PhaC1Psand PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 andphaDPs), and the gene order was ORF1,phaC1Ps,phaZPs,phaC2Ps, andphaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants ofPseudomonas putidaandRalstonia eutropharevealed that PHB synthase and two PHA synthases ofPseudomonassp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.
IncP Plasmids Are Unusually Effective in Mediating Conjugation of
Escherichia coli
and
Saccharomyces cerevisiae
: Involvement of the Tra2 Mating System
Authors: Bates Steven , Cashmore Annette M. , Wilkins Brian M.
Published: December 2019

Mobilizable shuttle plasmids containing the origin-of-transfer (
oriT
) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from
Escherichia coli
to
Saccharomyces cerevisiae
. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10
−5
transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of
oriT
in
cis
and the provision in
trans
of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (
trbB
, -
C
, -
D
, -
E
, -
F
, -
G
, -
H
, -
I
, -
J
, and -
L
) required for plasmid transfer to
E. coli
K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus
traF
is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.
Involvement of Both Dockerin Subdomains in Assembly of the
Clostridium thermocellum
Cellulosome
Authors: Lytle Betsy , Wu J. H. David
Published: December 2019

Clostridium thermocellum
produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in
Escherichia coli
. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.
Transcriptional Regulation and Organization of the
dcuA
and
dcuB
Genes, Encoding Homologous Anaerobic C
4
-Dicarboxylate Transporters in
Escherichia coli
Authors: Golby Paul , Kelly David J. , Guest John R. , Andrews Simon C.
Published: December 2019

The
dcuA
and
dcuB
genes of
Escherichia coli
encode homologous proteins that appear to function as independent and mutually redundant C
4
-dicarboxylate transporters during anaerobiosis. The
dcuA
gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (
aspA
), while
dcuB
is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (
fumB
). To learn more about the respective roles of the
dcu
genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy
dcuA
- and
dcuB-lacZ
transcriptional fusions. The results show that
dcuA
is constitutively expressed whereas
dcuB
expression is highly regulated. The
dcuB
gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition,
dcuB
is strongly induced by C
4
-dicarboxylates, suggesting that
dcuB
is under the control of an uncharacterized C
4
-dicarboxylate-responsive gene regulator. Northern blotting confirmed that
dcuA
(and
aspA
) is expressed under both aerobic and anaerobic conditions and that
dcuB
(and
fumB
) is induced anaerobically. Major monocistronic transcripts were identified for
aspA
and
dcuA
, as well as a minor species possibly corresponding to an
aspA-dcuA
cotranscript. Five major transcripts were observed for
dcuB
and
fumB
: monocistronic transcripts for both
fumB
and
dcuB
; a
dcuB-fumB
cotranscript; and two transcripts, possibly corresponding to
dcuB-fumB
and
fumB
mRNA degradation products. Primer extension analysis revealed independent promoters for
aspA
,
dcuA
, and
dcuB
, but surprisingly no primer extension product could be detected for
fumB
. The expression of
dcuB
is entirely consistent with a primary role for DcuB in mediating C
4
-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.
Disruption of
PMR1
, Encoding a Ca
2+
-ATPase Homolog in
Yarrowia lipolytica
, Affects Secretion and Processing of Homologous and Heterologous Proteins
Authors: Sohn Young-Sun , Park Cheon Seok , Lee Sun-Bok , Ryu Dewey D. Y.
Published: December 2019

The
Yarrowia lipolytica PMR1
gene (
YlPMR1
) is a
Saccharomyces cerevisiae PMR1
homolog which encodes a putative secretory pathway Ca
2+
-ATPase. In this study, we investigated the effects of a
YlPMR1
disruption on the processing and secretion of native and foreign proteins in
Y. lipolytica
and found variable responses by the
YlPMR1
-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the
YlPMR1
-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the
YlPMR1
-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca
2+
-ATPase genes on the secretion of rice α-amylase was also observed in the
Y. lipolytica
and
S. cerevisiae PMR1
-disrupted mutants. Unlike rice α-amylase, the secretion of
Trichoderma reesei
endoglucanase I (EGI) was not influenced by the
YlPMR1
disruption. However, the secreted EGI from the
YlPMR1
-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the
YlPMR1
-disrupted mutant. Our results indicate that the effects of the
YlPMR1
disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.
Superimposition of TyrR Protein-Mediated Regulation on Osmoresponsive Transcription of
Escherichia coli proU
In Vivo
Authors: Gowrishankar J. , Pittard A. J.
Published: December 2019

Osmotic regulation of
proU
expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the ς
70
-controlled promoter of
proU
in
Escherichia coli
, we were able to demonstrate a superposed TyrR-mediated activation by
l
-phenylalanine (Phe), as well as repression by
l
-tyrosine, of
proU
expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of
proU
at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in
E. coli.
Identification and Characterization of the
Erwinia amylovora rpoS
Gene: RpoS Is Not Involved in Induction of Fireblight Disease Symptoms
Authors: Anderson M. , Pollitt C. E. , Roberts I. S. , Eastgate J. A.
Published: December 2019

The
Erwinia amylovora rpoS
gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, an
E. amylovora rpoS
mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.
Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe
Authors: Madison-Antenucci Susan , Steege Deborah A.
Published: December 2019

Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it. Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from
Escherichia coli
. The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs. Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V. The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation. The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, ∼75-fold. As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred.
Toxicity of Linoleic Acid Hydroperoxide to
Saccharomyces cerevisiae
: Involvement of a Respiration-Related Process for Maximal Sensitivity and Adaptive Response
Authors: Evans Marguerite V. , Turton Hal E. , Grant Chris M. , Dawes Ian W.
Published: December 2019

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type
Saccharomyces cerevisiae
at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (
gsh1
and
gsh2
), glutathione reductase (
glr1
), respiratory competence ([
rho
0
] petite), and yAP-1p-mediated expression (
yap1
) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [
rho
0
] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the
yap1
and
glr1
mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 μg ml
−1
). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and
tert
-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37°C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.
Immunolocalization of Hsp60 in
Legionella pneumophila
Authors: Garduño Rafael A. , Faulkner Gary , Trevors Mary A. , Vats Neeraj , Hoffman Paul S.
Published: December 2019

One of the most abundant proteins synthesized by
Legionella pneumophila
, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including
L. pneumophila
, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in
L. pneumophila
grown in vitro, or in vivo inside of HeLa cells. For comparative purposes
Escherichia coli
, expressing recombinant
L. pneumophila
Hsp60, was employed. In contrast to
E. coli
, where Hsp60 was localized exclusively in the cytoplasm, in
L. pneumophila
Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked
L. pneumophila
organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by
L. pneumophila
was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in
L. pneumophila
and further suggest both the existence of a novel secretion mechanism (not present in
E. coli
) and a potential role in pathogenesis.
Demonstration of the Key Role Played by the PufX Protein in the Functional and Structural Organization of Native and Hybrid Bacterial Photosynthetic Core Complexes
Authors: Fulcher Timothy K. , Beatty J. Thomas , Jones Michael R.
Published: December 2019

The role of a component of the bacterial photosystem, the PufX protein, was examined by heterologous expression of the
pufX
gene from
Rhodobacter capsulatus
in a strain of
R. sphaeroides
that lacks the native
pufX
gene. The strain of
R. sphaeroides
containing the
R. capsulatus
PufX protein was capable of efficient transduction of light energy despite a low degree of sequence conservation between the PufX proteins from the two species. The organization of the hybrid reaction center/LH1 photosystem in strains of
R. sphaeroides
containing the
R. capsulatus
LH1 antenna complex was affected differently by the
R. sphaeroides
and
R. capsulatus
PufX proteins. We discuss the implications of our findings for the role of the PufX protein in organizing the bacterial photosystem for efficient transduction of light energy.
Expression and Characterization of (
R
)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by
Aeromonas caviae
Authors: Fukui Toshiaki , Shiomi Naofumi , Doi Yoshiharu
Published: December 2019

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of
Aeromonas caviae
proved that ORF3 in the
pha
locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as

phaJ
Ac
. Escherichia coli

BL21(DE3) carrying

phaJ
Ac

under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of

phaJ
Ac

except for the initial Met residue. The enoyl-CoA hydratase encoded by

phaJ
Ac

exhibited (
R
)-specific hydration activity toward
trans
-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (
R
)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of

phaJ
Ac

is a channeling pathway for supplying (
R
)-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate-
co
-3-hydroxyhexanoate) biosynthesis in
A. caviae.
A New Transformation-Deficient Mutant of
Haemophilus influenzae
Rd with Normal DNA Uptake
Authors: Gwinn M. L. , Ramanathan R. , Smith H. O. , Tomb J.-F.
Published: December 2019

Haemophilus influenzae
Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type
H. influenzae
and whose phage recombination is 8 times lower was isolated. The affected gene,
comM
, is induced during competence development in wild-type
H. influenzae
but not in RJ248.
Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog
Authors: Cunningham Adam F. , Spreadbury Claire L.
Published: December 2019

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How
Mycobacterium tuberculosis
survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy.
Mycobacterium bovis
BCG and
M. tuberculosis
H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured
Mycobacterium smegmatis
. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in
M. bovis
BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of
M. tuberculosis.
Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in
Saccharomyces cerevisiae
Authors: Wang Xinping , Mann Craig J. , Bai Yinlin , Ni Li , Weiner Henry
Published: December 2019

The full-length DNAs for two
Saccharomyces cerevisiae
aldehyde dehydrogenase (ALDH) genes were cloned and expressed in
Escherichia coli
. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K
+
ions. In contrast, the activity of ALDH5 increased in the presence of K
+
ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δ
ald1
and Δ
ald2
strains on ethanol was marginally slower than that of the parent strain. The Δ
ald1 Δald2
double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.
Biochemical and Genetic Characterization of
trans
-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading
Nocardioides
Strain
Authors: Iwabuchi Tokuro , Harayama Shigeaki
Published: December 2019

trans
-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium,
Nocardioides
sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The

K
m

and
k
cat values of this enzyme for
trans
-2′-carboxybenzalpyruvate were 50 μM and 13 s
−1
, respectively.
trans
-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of
trans
-2′-hydroxybenzalpyruvate hydratase-aldolases from
Pseudomonas putida
PpG7 and
Pseudomonas
sp. strain C18.
Mismatch Repair in
Escherichia coli
Cells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ
Authors: Harris Reuben S. , Ross Kimberly J. , Lombardo Mary-Jane , Rosenberg Susan M.
Published: December 2019

In vitro, the methyl-directed mismatch repair system of
Escherichia coli
requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.
Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of
Alcaligenes eutrophus
Authors: Massanz Christian , Schmidt Silke , Friedrich Bärbel
Published: December 2019

The cytoplasmic, NAD-reducing hydrogenase (SH) of
Alcaligenes eutrophus
H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most of
hoxF
,
hoxU
, and
hoxY
led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of
hoxF
and
hoxU
, displayed H
2
-dependent dye-reducing activity, the monomeric form did not mediate the activation of H
2
, although nickel was incorporated into HoxH. Deletion of
hoxH
and
hoxY
led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.
Importance of RpoS and Dps in Survival of Exposure of Both Exponential- and Stationary-Phase
Escherichia coli
Cells to the Electrophile
N
-Ethylmaleimide
Authors: Ferguson G. P. , Creighton R. I. , Nikolaev Y. , Booth I. R.
Published: December 2019

The mechanisms by which
Escherichia coli
cells survive exposure to the toxic electrophile
N
-ethylmaleimide (NEM) have been investigated. Stationary-phase
E. coli
cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing
E. coli
strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting
E. coli
cells against NEM.
Genetic and Functional Analysis of the Styrene Catabolic Cluster of
Pseudomonas
sp. Strain Y2
Authors: Velasco Ana , Alonso Sergio , García José L. , Perera J. , Díaz Eduardo
Published: December 2019

The chromosomal region of
Pseudomonas
sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes,
styABCD
, and two regulatory genes,
stySR
, were identified. This gene cluster when transferred to
Escherichia coli
W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source. Genes
styABCD
are homologous to those encoding the styrene upper catabolic pathway in
Pseudomonas fluorescens
ST. Northern blot analyses have confirmed that genes
styABCD
constitute a transcription unit. The transcription start site of the
sty
operon was mapped 33 nucleotides upstream of the
styA
translational start codon. The
styS
and
styR
genes, which form an independent transcriptional unit, are located upstream of the
styABCD
operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The
styS
gene product is homologous to histidine kinase proteins, whereas the
styR
gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains. Expression of the catabolic operon decreased significantly in the absence of the
stySR
genes and was restored when the
stySR
genes were provided in
trans
in the presence of styrene, suggesting that the
stySR
system behaves as a styrene-inducible positive regulator of the
styABCD
operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the
styS
gene. This activity was found to be induced in
Pseudomonas
sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of
Pseudomonas
sp. strain Y2.
Helicobacter pylori porCDABandoorDABCGenes Encode Distinct Pyruvate:Flavodoxin and 2-Oxoglutarate:Acceptor Oxidoreductases Which Mediate Electron Transport to NADP
Authors: Hughes Nicky J. , Clayton Chris L. , Chalk Peter A. , Kelly David J.
Published: December 2019

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953–3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype ofH. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. TheH. pylori por,oor, andfldAgenes were cloned and sequenced. The deducedporgene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophileHalobacterium halobium. BothporDandoorDencode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes inH. pylori, as insertion inactivation ofporBandoorAappeared to be lethal.
Regulation of Proteolysis of the Stationary-Phase Sigma Factor RpoS
Authors: Zhou Yanning , Gottesman Susan
Published: December 2019

RpoS, the stationary-phase sigma factor of
Escherichia coli
, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation in
clpP
, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.
Genetic Analysis of the
nuo
Locus, Which Encodes the Proton-Translocating NADH Dehydrogenase in
Escherichia coli
Authors: Falk-Krzesinski Holly J. , Wolfe Alan J.
Published: December 2019

Complex I (EC
1.6.99.3
) of the bacterium
Escherichia coli
is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the
E. coli
enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which
E. coli
cells regulate the expression of
nuo
and the assembly of complex I, we undertook a genetic analysis of the
nuo
locus, which encodes the 14 Nuo subunits comprising
E. coli
complex I. Here we present the results of studies, performed on an isogenic collection of
nuo
mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of
nuo
expression and/or the assembly of complex I.
Enzyme Specificity of 2-Nitrotoluene 2,3-Dioxygenase from
Pseudomonas
sp. Strain JS42 Is Determined by the C-Terminal Region of the α Subunit of the Oxygenase Component
Authors: Parales Juanito V. , Parales Rebecca E. , Resnick Sol M. , Gibson David T.
Published: December 2019

Biotransformations with recombinant
Escherichia coli
expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from
Pseudomonas
sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from
Burkholderia
sp. strain DNT (formerly
Pseudomonas
sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISPα) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISPβ) was shown to play no role in determining the specificities of these dioxygenases.
Identification of
dnaX
as a High-Copy Suppressor of the Conditional Lethal and Partition Phenotypes of the
parE10
Allele
Authors: Levine Cindy , Marians Kenneth J.
Published: December 2019

Termination of DNA replication, complete topological unlinking of the parental template DNA strands, partition of the daughter chromosomes, and cell division follow in an ordered and interdependent sequence during normal bacterial growth. In
Escherichia coli
, topoisomerase IV (Topo IV), encoded by
parE
and
parC
, is responsible for decatenation of the two newly formed chromosomes. In an effort to uncover the pathway of information flow between the macromolecular processes that describe these events, we identified
dnaX
, encoding the τ and γ subunits of the DNA polymerase III holoenzyme, as a high-copy suppressor of the temperature-sensitive phenotype of the
parE10
allele. We show that suppression derives from overexpression of the γ, but not the τ, subunit of the holoenzyme and that the partition defect of
parE10
cells is nearly completely reverted at the nonpermissive temperature as well. These observations suggest a possible association between Topo IV and the replication machinery.
HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in
Mycoplasma pneumoniae
Authors: Hahn Tae-Wook , Willby Melisa J. , Krause Duncan C.
Published: December 2019

Mycoplasma pneumoniae
proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in
hmw1
and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end of
p30
. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type
p30
allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type
M. pneumoniae
cells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant
p30
had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type
hmw1
allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an
hmw1
gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.
Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the
Escherichia coli
Phosphate Regulon
Authors: Haldimann Andreas , Daniels Larry L. , Wanner Barry L.
Published: December 2019

Escherichia coli
genes regulated by environmental inorganic phosphate (P
i
) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the P
i
-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated

P
araB

promoter or the rhamnose-regulated

P
rhaB

promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of

P
araB

or

P
rhaB

fusions and for recombination of them onto the
E. coli
chromosome at the
araCBAD
or
rhaRSBAD
locus, respectively. Using strains carrying such single-copy fusions, we showed that a

P
rhaB

fusion is more tightly regulated than a

P
araB

fusion in that a

P
rhaB
-phoR

+
fusion but not a

P
araB
-phoR

+
fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both

P
araB
-phoB

+
and

P
rhaB
-phoB

+
fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise Δ
phoU
mutations. However, we unexpectedly found that such Δ
phoU
mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797–6809, 1993). They also readily give rise to compensatory mutants with lesions in
phoB
,
phoR
, or a
pst
gene, making their study particularly difficult. Here we found that, by using

P
araB
-phoB

+
,

P
rhaB
-phoB

+
, or

P
rhaB
-phoR

+
fusions, we were able to overcome the extremely deleterious growth defect of a Pst
+
Δ
phoU
mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.
Localization of Cell Division Protein FtsK to the
Escherichia coli
Septum and Identification of a Potential N-Terminal Targeting Domain
Authors: Yu Xuan-chuan , Tran Anthony H. , Sun Qin , Margolin William
Published: December 2019

Escherichia coli
cell division protein FtsK is a homolog of
Bacillus subtilis
SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in
E. coli
cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the
ftsK44
mutation in
trans
, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of
ftsK44
, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and
ftsI
mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in
ftsZ
mutant filaments. In
ftsK44
mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the
ftsK44
mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.
Characterization of IS
1515
, a Functional Insertion Sequence in
Streptococcus pneumoniae
Authors: Muñoz Rosario , López Rubens , García Ernesto
Published: December 2019

We describe the characterization of a new insertion sequence, IS
1515
, identified in the genome of
Streptococcus pneumoniae
I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585–602, 1959). A copy of this element located in the
cap1E
I41R
gene was sequenced. The 871-bp-long IS
1515
element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS
1381
from
S. pneumoniae
, ISL
2
from
Lactobacillus helveticus
, IS
702
from the cyanobacterium
Calothrix
sp. strain PCC 7601, and IS
112
from
Streptomyces albus
G. IS
1515
appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain I41R is the result of both the presence of an IS
1515
copy and a frameshift mutation in the
cap1E
I41R
gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS
1515
behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS
1515
were also able to excise and move to another locations in the chromosome of
S. pneumoniae
. To our knowledge, this is the first report of a functional IS in pneumococcus.
Loss of the Plasma Membrane-Bound Protein Gas1p in
Saccharomyces cerevisiae
Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity
Authors: Ram Arthur F. J. , Kapteyn Johan C. , Montijn Roy C. , Caro L. Heleen P. , Douwes Jeroen E. , Baginsky Walter , Mazur Paul , van den Ende Herman , Klis Frans M.
Published: December 2019

Deletion of
GAS1/GGP1/CWH52
results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that
gas1
Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.