bluuebunny/crossref_metadata_2025 huggingface dataset:
Endonuclease V (
nfi
) Mutant of
Escherichia coli
K-12
Authors: Guo Guangming , Weiss Bernard
Published: December 2019

Endonuclease V (deoxyinosine 3′ endonuclease), the product of the
nfi
gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways,
nfi
insertion mutants and overproducers (strains bearing
nfi
plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An
nfi
mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an
nfi
mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A · T base pairs. An
nfi
mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or φX174 nor of Uracil-containing bacteriophage λ. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.
Cloning of Genes Coding for the Three Subunits of Thiocyanate Hydrolase of
Thiobacillus thioparus
THI 115 and Their Evolutionary Relationships to Nitrile Hydratase
Authors: Katayama Yoko , Matsushita Yasuhiko , Kaneko Miyuki , Kondo Mai , Mizuno Tadayoshi , Nyunoya Hiroshi
Published: December 2019

Thiocyanate hydrolase is a newly found enzyme from
Thiobacillus thioparus
THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170–9175, 1992). We have cloned and sequenced the
scn
genes that encode the three subunits of the enzyme. The
scnB
,
scnA
, and
scnC
genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the β, α, and γ subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the β and α subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the β subunit of nitrile hydratase, and the γ subunit of thiocyanate hydrolase was homologous to the α subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses.
Regulation of the Carnitine Pathway in
Escherichia coli
: Investigation of the
cai-fix
Divergent Promoter Region
Authors: Buchet Anne , Eichler Knut , Mandrand-Berthelot Marie-Andrée
Published: December 2019

The divergent structural operons
caiTABCDE
and
fixABCX
of
Escherichia coli
are required for anaerobic carnitine metabolism. Transcriptional monocopy
lacZ
fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an
fnr
mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the
cai
transcriptional start site, plays a direct role in the sole
cai
activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the
fix
promoter, caused only a threefold reduction in
fix
expression. Thus, the role of the third CRP site, located at −126.5 of
fix
, might be to reinforce the action of site 2. A critical 50-bp
cis
-acting sequence overlapping the
fix
mRNA start site was found, by deletion analysis, to be necessary for
cai
transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.
Genetic Instability of the Global Regulator
agr
Explains the Phenotype of the
xpr
Mutation in
Staphylococcus aureus
KSI9051
Authors: McNamara Peter J. , Iandolo John J.
Published: December 2019

Staphylococcus aureus
KSI9051 has a complex mutation that was associated with the aberrant expression of cell surface and extracellular proteins (M. S. Smeltzer, M. E. Hart, and J. J. Iandolo, J. Bacteriol. 61:919–925, 1993). This mutation was named
xpr
, although no specific gene was identified. Here this mutation is referred to as Δ1058::Tn
551
. In this study, we show that in strain KSI9051, the Δ1058::Tn
551
mutation occurred coincidentally with a frameshift in
agrC
that is expected to truncate the sensor component of the known staphylococcal global regulatory locus
agr
. Remarkably, pleiotropic mutations affecting cell surface and extracellular proteins are generated at frequencies approaching 50% upon the transduction of erythromycin resistance (Em
r
) encoded by Δ1058::Tn
551
from
S. aureus
KSI905 back to its parental strain, S6C. Three independent isolates created in the manner of KSI9051 contained mutations within
agrC
. Each isolate had different mutations, suggesting that the transduction of Em
r
encoded by Δ1058::Tn
551
affects the stability of
agrC
in S6C. In similar experiments with strains from an
S. aureus
8325 genetic background, a mutant AgrC phenotype could not be isolated, implying that strain S6 has aberrant genetic behavior. A comparison of the nucleotide sequences of AgrC from several strains revealed seven errors in the GenBank entry for
agr
(
X52543
); these data were confirmed with plasmid pRN6650, the original wild-type clone of
agr.
Isolation of a Periplasmic Molecular Chaperone-Like Protein of
Rhodobacter sphaeroides
f. sp.
denitrificans
That Is Homologous to the Dipeptide Transport Protein DppA of
Escherichia coli
Authors: Matsuzaki Masahiro , Kiso Yuso , Yamamoto Isamu , Satoh Toshio
Published: December 2019

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph
Rhodobacter sphaeroides
f. sp.
denitrificans
, in a manner similar to that of the
Escherichia coli
chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of
E. coli
. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.
Function of Protonatable Residues in the Flagellar Motor of
Escherichia coli
: a Critical Role for Asp 32 of MotB
Authors: Zhou Jiadong , Sharp Leslie L. , Tang H. Lucy , Lloyd Scott A. , Billings Stephanie , Braun Timothy F. , Blair David F.
Published: December 2019

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.
Role of Alternative ς Factor AlgU in Encystment of
Azotobacter vinelandii
Authors: Moreno Soledad , Nájera Rebeca , Guzmán Josefina , Soberón-Chávez Gloria , Espín Guadalupe
Published: December 2019

Alginate is essential for encystment in
Azotobacter vinelandii
. Transcription of the
algD
gene, which codes for GDP-mannose dehydrogenase, a key enzyme in the alginate biosynthetic pathway, is initiated at two promoters, one of which, p2, has ς
E
consensus sequences. AlgU is the
A. vinelandii
alternative ς
E
factor. In this study, we constructed an
algU
mutant (SMU88) which, as expected, is impaired in alginate production, encystment, and transcription of the
algD
gene from the p2 promoter. Plasmid pJMSAT1, carrying the
A. vinelandii algU
gene, restored alginate production and encystment to SMU88 and to strain UW136, a naturally occurring
algU
mutant. Plasmid pSMU865, carrying the
A. vinelandii mucABCD
genes coding for negative regulators of AlgU activity and previously shown to diminish alginate production in the wild-type strain, ATCC 9046, was shown here to impair encystment and transcription of the
algD
gene from the p2
algU
-dependent promoter. Since nonencysting strain ATCC 9046/pSMU865 produced more alginate than some encysting strains, such as UW136/pJMSAT1, we propose an AlgU role in encystment, independent of the structural role that alginate plays in mature cysts.
Expression of the Transposase Gene
tnpA
of Tn
4652
Is Positively Affected by Integration Host Factor
Authors: Hõrak Rita , Kivisaar Maia
Published: December 2019

Tn
4652
is a derivative of the toluene degradation transposon Tn
4651
that belongs to the Tn
3
family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270–276, 1987). We have sequenced the transposase gene
tnpA
of transposon Tn
4652
and mapped its promoter to the right end of the element. The deduced amino acid sequence of
tnpA
revealed 96.2% identity with the putative transposase of Tn
5041
. Homology with other Tn
3
family transposases was only moderate (about 20 to 24% identity), suggesting that Tn
4652
and Tn
5041
are distantly related members of the Tn
3
family. Functional analysis of the
tnpA
promoter revealed that it is active in
Pseudomonas putida
but silent in
Escherichia coli
, indicating that some
P. putida
-specific factor is required for the transcription from this promoter. Additionally,
tnpA
promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the
tnpA
promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the
P. putida ihfA
-deficient strain A8759. Moreover, the Tn
4652
terminal sequences had a negative effect on transcription from the
tnpA
promoter in the
ihfA
-defective strain. This finding suggests that IHF not only enhances transcription from the
tnpA
promoter but also alleviates the negative effect of terminal sequences of Tn
4652
on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn
4652
bind IHF from a cell lysate of
E. coli.
Effects of Iron and Temperature on Expression of the
Pseudomonas aeruginosa tolQRA
Genes: Role of the Ferric Uptake Regulator
Authors: Lafontaine Eric R. , Sokol Pamela A.
Published: December 2019

The
tolQRA
genes have been recently identified in
Pseudomonas aeruginosa
PAO. In this study, we examined the effect of iron and temperature on
tolQRA
expression. A promoterless
lacZ
gene was introduced downstream of plasmid-encoded
tolQ
and
tolA
, and expression was monitored by measuring β-galactosidase activity of cultures. Addition of 25 μM FeCl
3
to the culture medium reduced
tolQRA
expression by 50 to 60% in PAO but by only 25% in the
fur
mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the
P. aeruginosa
ferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site of
tolA
. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to the
tolA
or
tolQR
promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of the
tol
genes appears to involve an intermediate regulatory gene. Expression of
tolQR
and
tolA
was optimal at 37°C and was reduced by 40 to 50% when cultures were grown at either 42 or 25°C. Growth in high-iron medium at 25°C further reduced
tolQR
and
tolA
expression.
Identification and Characterization of
MAE1
, the
Saccharomyces cerevisiae
Structural Gene Encoding Mitochondrial Malic Enzyme
Authors: Boles Eckhard , de Jong-Gubbels Patricia , Pronk Jack T.
Published: December 2019

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless,
pyk1 pyk2
mutants of
Saccharomyces cerevisiae
devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame
YKL029c
, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of
YKL029c/MAE1
from the
MET25
promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of
MAE1
alone did not result in a clear phenotype. Regulation of
MAE1
was studied by determining enzyme activities and
MAE1
mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a
MAE1
::
lacZ
fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,
MAE1
was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in
S. cerevisiae
is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.
d-Alanylcardiolipin, a Major Component of the Unique Lipid Pattern ofVagococcus fluvialis
Authors: Fischer Werner , Arneth-Seifert Doris
Published: December 2019

Motile group N streptococci, classified asVagococcus fluvialis, have been isolated from cows’ udders, human and animal feces, river water, and seawater. They possess an unusual membrane lipid and fatty acid pattern. We isolated and characterized 13 polar lipids, 8 of them also found in other gram-positive bacteria: mono- and dihexosyldiacylglycerol, an acylated and a glycerophosphate-substituted derivative of the latter, cardiolipin, phosphatidylglycerol,d-alanylphosphatidylglycerol, andl-lysylphosphatidylglycerol. Besides them, we characterized two rare compounds, bis(acylglycero)phosphate and α-d-glucopyranosylcardiolipin, and two compounds so far not detected in nature,d-alanylbis(acylglycero)phosphate andd-alanylcardiolipin. The concomitant occurrence of four aminoacyl phospholipids in one organism is another unique finding. Substituted cardiolipins represent a novel lipid class: in vagococci,d-alanylcardiolipin is a major membrane lipid component, contributing 11 and 26 mol% of total lipids in the exponential and stationary phases of growth, respectively. The vagococcal lipids contain even-numbered straight-chain saturated andcis-monounsaturated fatty acids, but thecis-monoenic acids belong to the ω-9 series and not the ω-7 series, found in enterococci, lactococci, and streptococci.
Characterization of Chemotactic Responses and Flagella of
Hyphomicrobium
Strain W1-1B
Authors: Tuhela Laura , Robinson Jayne B. , Tuovinen Olli H.
Published: December 2019

Motile swarmer cells of
Hyphomicrobium
strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff’s staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of
Rhizobium meliloti
cross-reacted with the striated flagella of
Hyphomicrobium
strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.
Analysis of
cis
-Acting Elements Required for
bfpA
Expression in Enteropathogenic
Escherichia coli
Authors: Bustamante Victor H. , Calva Edmundo , Puente Jose Luis
Published: December 2019

bfpA
expression in enteropathogenic
Escherichia coli
is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators. Site-directed and PCR random mutagenesis, as well as deletion analysis of the
bfpA
upstream regulatory region, supported assignment of the promoter elements and demonstrated that the
cis
-acting elements that mediate BfpT-dependent regulation of
bfpA
are located between positions −85 and −46. Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the
bfpT
gene, which is essential for
bfpT
autoregulation.
Changes in Ribosomal Activity of
Escherichia coli
Cells during Prolonged Culture in Sea Salts Medium
Authors: Kalpaxis Dimitrios L. , Karahalios Panagiotis , Papapetropoulou M.
Published: December 2019

The activity of ribosomes from a clinical isolate of
Escherichia coli
, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in the
k
max
/

K
s

ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the
k
max
/

K
s

value. {
k
max
is the apparent catalytic rate constant of peptidyl transferase, and

K
s

is the dissociation constant of the encounter complex between acetyl (Ac)[
3
H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.
OpaR, a Homolog of
Vibrio harveyi
LuxR, Controls Opacity of
Vibrio parahaemolyticus
Authors: McCarter Linda L.
Published: December 2019

Vibrio parahaemolyticus
is an organism well adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces.
V. parahaemolyticus
displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types. Although not itself luminescent,
V. parahaemolyticus
produces autoinducer molecules capable of inducing luminescence in
Vibrio harveyi
. To examine the role of quorum signaling in the lifestyles of
V. parahaemolyticus
, the functional homolog of the gene encoding the
V. harveyi
autoinducer-controlled transcriptional regulatory protein LuxR was cloned. Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR. Introduction of the clone carrying the
luxR
-like locus into
V. parahaemolyticus
dramatically affected colony morphology, converting a translucent strain to an opaque one. When the coding sequence for the
luxR
homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-β-
d
-thiogalactopyranoside. Allelic disruption of the
luxR
-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability. Primer extension mapping demonstrated
opaR
transcription in opaque but not translucent cell types. It is postulated that this gene, which has been named
opaR
, encodes a transcription factor controlling cell type. The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the
opaR
locus of opaque and translucent strains.
Multiple Transcriptional Control of the
Lactococcus lactis trp
Operon
Authors: Raya Raul , Bardowski Jacek , Andersen Paal S. , Ehrlich S. Dusko , Chopin Alain
Published: December 2019

The
Lactococcus lactis trpEGDCFBA
operon is preceded by a noncoding leader region. Transcriptional studies of the
trp
operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique P

trp

promoter, transcription termination at either T1 (upstream of the
trp
operon) or T2 (downstream of the
trp
operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.
Cloning, Sequencing, and Characterization of the
recA
Gene from
Rhodopseudomonas viridis
and Construction of a
recA
Strain
Authors: Chen I-Peng , Michel Hartmut
Published: December 2019

A recombination-deficient strain of the phototrophic bacterium
Rhodopseudomonas viridis
was constructed for the homologous expression of modified photosynthetic reaction center genes. The
R. viridis recA
gene was cloned and subsequently deleted from the
R. viridis
genome. The cloned
R. viridis recA
gene shows high identity to known
recA
genes and was able to complement the Rec
−
phenotype of a
Rhizobium meliloti recA
strain. The constructed
R. viridis recA
strain showed the general Rec
−
phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.
Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine
Authors: Rangaswamy Vidhya , Mitchell Robin , Ullrich Matthias , Bender Carol
Published: December 2019

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium
Pseudomonas syringae
. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated
cfa8
,
cfa9
, and
tnp1
; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of
cfa8
and
cfa9
were overproduced in
Escherichia coli
BL21; however,
tnp1
was not translated in these experiments. Mutagenesis and complementation analysis indicated that
cfa8
is required for the production of CFA and COR. Analysis of a
cfa9
mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway;
tnp1
, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a
P. syringae
strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.
The V Antigen of
Yersinia pestis
Regulates Yop Vectorial Targeting as Well as Yop Secretion through Effects on YopB and LcrG
Authors: Nilles Matthew L. , Fields Kenneth A. , Straley Susan C.
Published: December 2019

Yersinia pestis
expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca
2+
concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca
2+
as part of the low-Ca
2+
response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of
lcrV
and
yop
operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed
lcrG
deletion strain, a Δ
lcrG2
mutant, of
Y. pestis
that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca
2+
and to dramatically decrease Yop expression and secretion in growth medium lacking Ca
2+
. Overexpression of both LcrG and LcrV in the Δ
lcrG2
strain restored wild-type levels of Yop expression and Ca
2+
control of Yop secretion. Surprisingly, when HeLa cells were infected with the Δ
lcrG2
strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV’s regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.
Evolution of Cyanobacteria by Exchange of Genetic Material among Phyletically Related Strains
Authors: Rudi Knut , Skulberg Olav M. , Jakobsen Kjetill S.
Published: December 2019

The cyanobacterial radiation consists of several lineages of phyletically (morphologically and genetically) related organisms. Several of these organisms show a striking resemblance to fossil counterparts. To investigate the molecular mechanisms responsible for stabilizing or homogenizing cyanobacterial characters, we compared the evolutionary rates and phylogenetic origins of the small-subunit rRNA-encoding DNA (16S rDNA), the conserved gene
rbcL
(encoding
d
-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit), and the less conserved gene
rbcX
. This survey includes four categories of phyletically related organisms: 16 strains of
Microcystis
, 6 strains of
Tychonema
, 10 strains of
Planktothrix
, and 12 strains of
Nostoc
. Both
rbcL
and
rbcX
can be regarded as neutrally evolving genes, with 95 to 100% and 50 to 80% synonymous nucleotide substitutions, respectively. There is generally low sequence divergence within the
Microcystis
,
Tychonema
, and
Planktothrix
categories both for
rbcLX
and 16S rDNA. The
Nostoc
category, on the other hand, consists of three genetically clustered lineages for these loci. The 16S rDNA and
rbcLX
phylogenies are not congruent for strains within the clustered groups. Furthermore, analysis of the phyletic structure for
rbcLX
indicates recombinational events between the informative sites within this locus. Thus, our results are best explained by a model involving both intergenic and intragenic recombinations. This evolutionary model explains the DNA sequence clustering for the modern species as a result of sequence homogenization (concerted evolution) caused by exchange of genetic material for neutrally evolving genes. The morphological clustering, on the other hand, is explained by structural and functional stability of these characters. We also suggest that exchange of genetic material for neutrally evolving genes may explain the apparent stability of cyanobacterial morphological characters, perhaps over billions of years.
Secretion of Elastinolytic Enzymes and Their Propeptides by
Pseudomonas aeruginosa
Authors: Braun Peter , de Groot Arjan , Bitter Wilbert , Tommassen Jan
Published: December 2019

Elastase of
Pseudomonas aeruginosa
is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a
Streptomyces griseus
aminopeptidase accumulated under these conditions.
Identification of the Repressor-Encoding Gene of the
Lactobacillus
Bacteriophage A2
Authors: Ladero Victor , García Pilar , Bascarán Victoria , Herrero Mónica , Alvarez Miguel A. , Suárez Juan E.
Published: December 2019

The repressor gene of the
Lactobacillus
phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the λ Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected.
Aberrant Cell Division and Random FtsZ Ring Positioning in
Escherichia coli cpxA
* Mutants
Authors: Pogliano Joe , Dong Jian Ming , De Wulf Peter , Furlong Dierdre , Boyd Dana , Losick Richard , Pogliano Kit , Lin E. C. C.
Published: December 2019

In
Escherichia coli
, certain mutations in the
cpxA
gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the
cpxRA
operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.
Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet
Authors: Appleby Jeryl L. , Bourret Robert B.
Published: December 2019

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several
cheY
mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che
+
swarm phenotype. Although nonchemotactic,
cheY
mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.
Identification of a Gene Product Induced by Hard-Surface Contact of
Colletotrichum gloeosporioides
Conidia as a Ubiquitin-Conjugating Enzyme by Yeast Complementation
Authors: Liu Zhi-Mei , Kolattukudy Pappachan E.
Published: December 2019

The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host’s surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen
Colletotrichum gloeosporioides
only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes,
chip1
(
Colletotrichum
hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (
ubc1
Cg
) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the
Saccharomyces cerevisiae
UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1
Cg
can complement the proteolysis deficiency of the
S. cerevisiae ubc4 ubc5
mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.
Circadian Rhythm of Nitrogenase Gene Expression in the Diazotrophic Filamentous Nonheterocystous Cyanobacterium
Trichodesmium
sp. Strain IMS 101
Authors: Chen Yi-Bu , Dominic Benny , Mellon Mark T. , Zehr Jonathan P.
Published: December 2019

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium
Trichodesmium
sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in
Trichodesmium
sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5°C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.
Differential Regulation of
Rhizobium etli rpoN2
Gene Expression during Symbiosis and Free-Living Growth
Authors: Michiels Jan , Moris Martine , Dombrecht Bruno , Verreth Christel , Vanderleyden Jos
Published: December 2019

The
Rhizobium etli rpoN1
gene, encoding the alternative sigma factor ς
54
(RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D’hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729–1740, 1998). We identified a second
rpoN
gene copy in
R. etli
,
rpoN2
, encoding a 54.0-kDa protein which displays 59% amino acid identity with the
R. etli
RpoN1 protein. The
rpoN2
gene is cotranscribed with a short open reading frame,
orf180
, which codes for a protein with a size of 20.1 kDa that is homologous to several prokaryotic and eukaryotic proteins of similar size. In contrast to the
R. etli rpoN1
mutant strain, inactivation of the
rpoN2
gene did not produce any phenotypic defects during free-living growth. However, symbiotic nitrogen fixation was reduced by approximately 90% in the
rpoN2
mutant, whereas wild-type levels of nitrogen fixation were observed in the
rpoN1
mutant strain. Nitrogen fixation was completely abolished in the
rpoN1 rpoN2
double mutant. Expression of
rpoN1
was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. In contrast,
rpoN2-gusA
and
orf180-gusA
fusions were not expressed aerobically but were strongly induced at low oxygen tensions or in bacteroids. Expression of
rpoN2
and
orf180
was abolished in
R. etli rpoN1 rpoN2
and
nifA
mutants under all conditions tested. Under free-living microaerobic conditions, transcription of
rpoN2
and
orf180
required the RpoN1 protein. In symbiosis, expression of
rpoN2
and
orf180
occurred independently of the
rpoN1
gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.
The Central, Surface-Exposed Region of the Flagellar Hook Protein FlgE of
Campylobacter jejuni
Shows Hypervariability among Strains
Authors: Lüneberg Edeltraud , Glenn-Calvo Eduardo , Hartmann Maike , Bär Werner , Frosch Matthias
Published: December 2019

In a previous study, we observed that monoclonal antibodies raised against the hook protein FlgE of
Campylobacter jejuni
LIO 36, isolate 5226, bound exclusively to this strain. The aim of this study was to elucidate the molecular basis for these binding specificities. The hook protein-encoding gene
flgE
of
C. jejuni
was cloned in
Escherichia coli
and sequenced. The
flgE
genes of four additional
C. jejuni
strains were amplified by PCR and also sequenced. Comparison of the deduced amino acid sequences revealed a high degree of variability in the central parts of the FlgE proteins among the strains, including variable and hypervariable domains. These findings may indicate a selective pressure of
C. jejuni
hosts, forcing the bacteria to generate variations in surface-exposed antigenic determinants.
Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in
Synechocystis
sp. Strain PCC6803: Effect of
pxcA
Inactivation on CO
2
, HCO
3
−
, and NO
3
−
Uptake
Authors: Sonoda Masatoshi , Katoh Hirokazu , Vermaas Wim , Schmetterer George , Ogawa Teruo
Published: December 2019

The product of
pxcA
(formerly known as
cotA
) is involved in light-induced Na
+
-dependent proton extrusion. In the presence of 2,5-dimethyl-
p
-benzoquinone, net proton extrusion by
Synechocystis
sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO
2
fixation by glyceraldehyde in a cytochrome
c
oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO
2
fixation is inhibited. At pH 8.0, NO
3
−
uptake activity was very low in the
pxcA
mutant at low [Na
+
] (∼100 μM). At pH 6.5, the
pxcA
strain did not take up CO
2
or NO
3
−
at low [Na
+
] and showed very low CO
2
uptake activity even at 15 mM Na
+
. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO
2
, HCO
3
−
, and NO
3
−
is discussed.
Transcriptional Analysis of Different Promoters in the
sar
Locus in
Staphylococcus aureus
Authors: Manna Adhar C. , Bayer Manfred G. , Cheung Ambrose L.
Published: December 2019

The expression of extracellular virulence determinants in
Staphylococcus aureus
is controlled by a 510-nucleotide RNA molecule (RNAIII) which is a part of the
agr
system. The
agr
operon, which encodes a multicomponent signal transduction system, is partially under the influence of an unlinked regulatory locus called
sar
. The
sar
locus is composed of three overlapping transcripts, designated
sarA
(0.56 kb),
sarC
(0.8 kb), and
sarB
(1.2 kb), originating from the P1, P3, and P2 promoters, respectively. In this study, we analyzed the differential expression of these promoters by using transcriptional fusion with the
xylE
reporter gene to study the activation of the
sar
locus. The data confirm the existence of three independent promoters with different promoter activities. Maximal promoter activity was observed with the combined fusion of P
2
-P
3
-P
1
promoters. Expression studies with a
sigB
mutant revealed that the P3 promoter is SigB dependent. Analysis of these transcriptional fusions in a
sarA
mutant and in complemented strains with each of the
sar
transcriptional units revealed that the
sar
locus is autoregulatory, with SarA acting as a positive regulator. From various transcriptional fusion studies of the upstream region of the P1 promoter, we have localized a 34-bp sequence which seems to play a role in down-modulating P1 transcription. Using heparin-Sepharose and DNA-specific columns, we partially purified a 12-kDa protein, possibly a repressor, which binds to the promoter regions upstream of P2 and P1 and which also binds to the 34-bp sequence. These data indicated that the regulation of the
sar
locus is complex and may involve the
sar
gene product(s) and other regulatory protein(s).
YscO of
Yersinia pestis
Is a Mobile Core Component of the Yop Secretion System
Authors: Payne Patricia L. , Straley Susan C.
Published: December 2019

The
Yersinia pestis
low-Ca
2+
response stimulon is responsible for the temperature- and Ca
2+
-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that
lcrD
,
yscC
,
yscD
,
yscG
, and
yscR
encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca
2+
. In this study, we characterized
yscO
of the Yop secretion (
ysc
) operon that contains
yscN
through
yscU
by determining the localization of its gene product and the phenotype of an in-frame deletion. The
yscO
mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca
2+
. In the absence of Ca
2+
, the mutant grew independently of Ca
2+
, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing
yscO
in
trans
in the
yscO
mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the
yscO
gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca
2+
and in the culture medium in the absence of Ca
2+
. YscO is the only mobile protein identified so far in the
Yersinia
species that is required for secretion of V antigen and Yops.