bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of a Gene Encoding a Putative Glycoprotease Leads to Reduced Salt Tolerance, Altered Pigmentation, and Cyanophycin Accumulation in the CyanobacteriumSynechocystissp. Strain PCC 6803
Authors: Zuther Ellen , Schubert Hendrik , Hagemann Martin
Published: December 2019

The salt-sensitive mutant 549 of the cyanobacteriumSynechocystissp. strain PCC 6803 was genetically and physiologically characterized. The mutated site and corresponding wild-type site were cloned and partially sequenced. The genetic analysis revealed that during the mutation about 1.8 kb was deleted from the chromosome of mutant 549. This deletion affected four open reading frames: agcpgene homolog, thepsaFJgenes, and an unknown gene. After construction of mutants with single mutations, only thegcpmutant showed a reduction in salt tolerance comparable to that of the initial mutant, indicating that the deletion of this gene was responsible for the salt sensitivity and that the other genes were of minor importance. Besides the reduced salt tolerance, a remarkable change in pigmentation was observed that became more pronounced in salt-stressed cells. The phycobilipigment content decreased, and that of carotenoids increased. Investigations of changes in the ultrastructure revealed an increase in the amount of characteristic inclusion bodies containing the high-molecular-weight nitrogen storage polymer cyanophycin (polyaspartate and arginine). The salt-induced accumulation of cyanophycin was confirmed by chemical estimations. The putative glycoprotease encoded by thegcpgene might be responsible for the degradation of cyanophycin inSynechocystis. Mutation of this gene leads to nitrogen starvation of the cells, accompanied by characteristic changes in pigmentation, ultrastructure, and salt tolerance level.
Characterization of the Starvation-Survival Response of
Staphylococcus aureus
Authors: Watson Sean P. , Clements Mark O. , Foster Simon J.
Published: December 2019

The starvation-survival response of
Staphylococcus aureus
as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [
35
S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.
The TATA-Binding Protein (TBP) from the Human Fungal PathogenCandida albicansCan Complement Defects in Human and Yeast TBPs
Authors: Leng Ping , Carter Philip E. , Brown Alistair J. P.
Published: December 2019

Candida albicansis the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed theC. albicansTATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a singleC. albicans TBP1locus is expressed at similar levels in the yeast and hyphal forms of this fungus. TheTBP1open reading frame is 716 bp long and encodes a functional TBP of 27 kDa.C. albicansTBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15mutation inSaccharomyces cerevisiae. The predicted amino acid sequences of TBPs fromC. albicansand other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.
Positive and Negative Transcriptional Regulation of the
Escherichia coli
Gluconate Regulon Gene
gntT
by GntR and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Authors: Peekhaus Norbert , Conway T.
Published: December 2019

The
gntT
gene of
Escherichia coli
is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of
gntT
expression since gluconate is a catabolite-repressing sugar. In a
gntR
deletion mutant, the expression of a chromosomal
gntT
::
lacZ
fusion is both high and constitutive, confirming that GntR is the negative regulator of
gntT
. Indeed, GntR binds to two consensus
gnt
operator sites; one overlaps the −10 region of the
gntT
promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when
gntR
deletion strains are grown in the presence of gluconate, there is a twofold decrease in
gntT
expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of
gntR
mutants to the inducer is termed ultrarepression. Transcription of
gntT
is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the
gntT
transcription start site.
Characterization of a Novel Member of the DegS-DegU Regulon Affected by Salt Stress in
Bacillus subtilis
Authors: Dartois Véronique , Débarbouillé Michel , Kunst Frank , Rapoport Georges
Published: December 2019

As a soil bacterium also found in estuarine and marine habitats,
Bacillus subtilis
has evolved various sensing and adaptation systems in order to face salt stress conditions. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which was previously shown to be stimulated by high salt concentrations. A search for promoters regulated in response to salt stress led to the identification of
wapA
, encoding a wall-associated protein, which is strongly expressed at low salt concentrations and almost completely repressed in the presence of 0.7 M disodium succinate. Repression of
wapA
transcription by salt stress was shown to require the phosphorylated form of DegU. Moreover, DegU-mediated repression of
wapA
occurred only in high-salt medium. Alignment between the control region of
wapA
and other DegU-regulated promoters allowed the identification of a putative DegU target sequence, AGAAN
11
TTCAG. Mutation/deletion analyses of the
wapA
promoter region confirmed the role of the putative DegU control site in repression of
wapA
transcription at high salt concentrations and revealed a second site of repression located downstream from the transcription start site. Since residual negative control was observed at this second site in the absence of DegU, it seems likely that an additional repressor acts on the
wapA
control region to further downregulate
wapA
transcription under salt stress conditions.
Regulation of
ntcA
Expression and Nitrite Uptake in the Marine
Synechococcus
sp. Strain WH 7803
Authors: Lindell Debbie , Padan Etana , Post Anton F.
Published: December 2019

NtcA is a transcriptional activator involved in global nitrogen control in cyanobacteria. In the absence of ammonium it regulates the transcription of a series of genes encoding proteins required for the uptake and assimilation of alternative nitrogen sources (I. Luque, E. Flores, and A. Herrero, EMBO J. 13:2862–2869, 1994).
ntcA
, present in a single copy in the marine
Synechococcus
sp. strain WH 7803, was cloned and sequenced. The putative amino acid sequence shows a high degree of identity to NtcA from freshwater cyanobacteria in two functional domains. The expression of
ntcA
was negatively regulated by ammonium from a putative transcription start point located downstream of an NtcA consensus recognition sequence. Addition of either rifampin or ammonium led to a rapid decline in
ntcA
transcript levels with half-lives of less than 2 min in both cases. Nitrate-grown cells showed high
ntcA
transcript levels, as well as the capacity for active nitrite uptake. However, ammonium-grown cells showed low levels of the
ntcA
transcript and did not utilize nitrite. The addition of ammonium to nitrite uptake-active cells resulted in a gradual decline in the rate of uptake over a 24-h period. Active nitrite uptake was not induced in cells transferred to medium lacking a nitrogen source despite evidence of elevated expression of
ntcA
, indicating that
ntcA
expression is not sufficient for uptake capacity to develop. Nitrate and nitrite addition led to the development of nitrite uptake, whereas the addition of leucine did not. Furthermore, nitrite addition triggered the de novo protein synthesis required for uptake capacity to develop. These data suggest that nitrite and nitrate act as specific inducers for the synthesis of proteins required for nitrite uptake.
A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in
Lactococcus lactis
Authors: Le Loir Y. , Gruss A. , Ehrlich S. D. , Langella P.
Published: December 2019

Lactococcus lactis
, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of
L. lactis
by using the
Staphylococcus aureus
nuclease (Nuc) as a heterologous model protein. When expressed in
L. lactis
from an efficient lactococcal promoter and its native signal peptide, only ∼60% of total Nuc was present in a secreted form at ∼5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ∼30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ∼80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ∼20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase of
Bacillus stearothermophilus
, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in
L. lactis.
Sterol Uptake in
Saccharomyces cerevisiae
Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification
Authors: Ness Frédérique , Achstetter Tilman , Duport Catherine , Karst Francis , Spagnoli Roberto , Degryse Eric
Published: December 2019

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in
erg
auxotrophs.
Escherichia coli mrsC
Is an Allele of
hflB
, Encoding a Membrane-Associated ATPase and Protease That Is Required for mRNA Decay
Authors: Wang Rong-fu , O’Hara Eileen B. , Aldea Marti , Bargmann Cornelia I. , Gromley Heather , Kushner Sidney R.
Published: December 2019

The
mrsC
gene of
Escherichia coli
is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive
mrsC505
allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920–1928, 1998). The cloned
mrsC
gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain.
mrsC
is identical to the independently identified
ftsH
gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344–1351, 1993). The purified protein had a

K
m

of 28 μM for ATP and a
V
max
of 21.2 nmol/μg/min. An amino-terminal glutathione
S
-transferase–MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (
mrsC201
) abolished the complementation of the
mrsC505
mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, the
mrsC505
allele conferred a temperature-sensitive HflB phenotype, while the
hflB29
mutation promoted mRNA stability at both 30 and 44°C, suggesting that the inviability associated with the
mrsC505
allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in
E. coli.
The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of
Escherichia coli
Are Important but Not Essential for Cytochrome
c
Maturation In Vivo
Authors: Fabianek Renata A. , Hennecke Hauke , Thöny-Meyer Linda
Published: December 2019

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome
c
maturation in
Escherichia coli
. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in
ccmG
resulted in the complete absence of all
c
-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of
Bradyrhizobium japonicum
holocytochrome
c
550
expressed in
E. coli
. This defect, but not that of the
ccmG
null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.
Frequency of Pilin Antigenic Variation in
Neisseria gonorrhoeae
Authors: Serkin Carla D. , Seifert H. Steven
Published: December 2019

Variation of the pilus of
Neisseria gonorrhoeae
occurs by the recombination of silent pilin DNA sequences into the pilin expression locus. We have developed a quantitative, competitive reverse transcription-PCR assay which measures the frequency of pilin antigenic variation independently of changes in gonococcal colony morphology and have determined this frequency within a gonococcal population. We have also studied the frequency of antigenic variation during growth and have concluded that growth does not dramatically influence the frequency of pilin antigenic variation, although a reproducible, twofold increase is observed upon the transition into late log/stationary phase.
Occurrence of Homologs of the
Escherichia coli lytB
Gene in Gram-Negative Bacterial Species
Authors: Potter Sheila , Yang Xiaoming , Boulanger Martin J. , Ishiguro Edward E.
Published: December 2019

The
Escherichia coli
LytB protein regulates the activity of guanosine 3′,5′-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the
E. coli lytB
gene as a probe revealed the presence of
lytB
homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The
lytB
homologs from
Enterobacter aerogenes
and
Pseudomonas fluorescens
complemented the
E. coli lytB44
mutant allele.
Growth Rate-Dependent Accumulation of RNA from Plasmid-Borne rRNA Operons in
Escherichia coli
Authors: Stevenson Bradley S. , Schmidt Thomas M.
Published: December 2019

Inadequate regulation of the expression of additional plasmid-borne rRNA operons in
Escherichia coli
was exaggerated at slow growth rates, resulting in increases of approximately 100% for RNA concentration and 33% for doubling time. These observations are consistent with the hypothesis that multiple rRNA operons constitute a metabolic burden at slow growth rates.
Enterocins L50A and L50B, Two Novel Bacteriocins from
Enterococcus faecium
L50, Are Related to Staphylococcal Hemolysins
Authors: Cintas Luis M. , Casaus Pilar , Holo Helge , Hernandez Pablo E. , Nes Ingolf F. , Håvarstein Leiv Sigve
Published: December 2019

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodrı́guez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643–2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by
Enterococcus faecium
L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that
entL50A
and
entL50B
are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.
Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69
Authors: Yeh Lu-Shu , Hsu Tien , Karam Jim D.
Published: December 2019

The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene
46-45.2-
(
rpbA
)
-45-44-62
cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the
rpbA
region (which encodes a nonessential protein in T4) is highly diverged (∼49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.
Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism
Authors: Jung Kwang-Hwan , Spudich John L.
Published: December 2019

The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon
Halobacterium salinarum
. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (
K
eq
) for the conformational transition in different mutants and mutant-suppressor pairs.
The
katX
Gene, Which Codes for the Catalase in Spores of
Bacillus subtilis
, Is a Forespore-Specific Gene Controlled by ς
F
, and KatX Is Essential for Hydrogen Peroxide Resistance of the Germinating Spore
Authors: Bagyan Irina , Casillas-Martinez Lilliam , Setlow Peter
Published: December 2019

Previous work has shown that the
katX
gene encodes the major catalase in dormant spores of
Bacillus subtilis
but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a
katX-lacZ
fusion began at approximately h 2 of sporulation, and >75% of the
katX
-driven β-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor ς
F
abolished
katX-lacZ
expression, while mutations in genes encoding ς
E
, ς
G
, and ς
K
did not. Induction of ς
F
synthesis in vegetative cells also resulted in
katX-lacZ
expression, while induction of ς
G
expression did not; the
katX-lacZ
fusion was also not induced by hydrogen peroxide. Upstream of the in vivo
katX
transcription start site there are sequences with good homology to those upstream of known ς
F
-dependent start sites. These data indicate that
katX
is an additional member of the forespore-specific ς
F
regulon. A mutant in the
katA
gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while a
katX
mutant was not. However, outgrowth of
katX
spores, but not
katA
spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.
Temperature Sensitivity of Bacteriolysis Induced by β-Lactam Antibiotics in Amino Acid-Deprived
Escherichia coli
Authors: Rodionov Dmitrii G. , Ishiguro Edward E.
Published: December 2019

The temperature-sensitive penicillin tolerance response previously reported in amino acid-deprived
Escherichia coli
(W. Kusser and E. E. Ishiguro, J. Bacteriol. 169:2310–2312, 1987) was not due to the induction of the heat shock response resulting from a temperature upshift and was therefore unrelated to the findings of another report (J. K. Powell and K. D. Young, J. Bacteriol. 173:4021–4026, 1991) indicating a positive correlation between the expression of heat shock proteins and penicillin tolerance. The thermosensitive event occurred in the lysis induction stage.
Specific Binding of
Escherichia coli
Ribosomal Protein S1 to
boxA
Transcriptional Antiterminator RNA
Authors: Mogridge Jeremy , Greenblatt Jack
Published: December 2019

We show that ribosomal protein S1 specifically binds the
boxA
transcriptional antiterminator RNAs of bacteriophage λ and the
Escherichia coli
ribosomal RNA operons. Although S1 competes with the NusB-S10 antitermination complex for binding to
boxA
, it does not affect antitermination by the λ N protein in vitro, and its role, if any, in rRNA synthesis is still unknown.
Physiological Regulation of the Derepressible Phosphate Transporter inSaccharomyces cerevisiae
Authors: Martinez Paula , Zvyagilskaya Renata , Allard Peter , Persson Bengt L.
Published: December 2019

The extracellular phosphate concentration permissive for the expression of different amounts of the active high-affinity Pho84 phosphate transporter in the plasma membrane as well as thePHO84messenger RNA levels in low-phosphate-grownSaccharomyces cerevisiaecells is very narrow and essential for a tight regulation of the transporter. The Pho84 transporter undergoes a rapid degradation once the supply of phosphate and/or carbon source is exhausted.
The
yhhP
Gene Encoding a Small Ubiquitous Protein Is Fundamental for Normal Cell Growth of
Escherichia coli
Authors: Yamashino Takafumi , Isomura Mototoshi , Ueguchi Chiharu , Mizuno Takeshi
Published: December 2019

H-NS is a major constituent of the
Escherichia coli
nucleoid, whereas ς
S
is a stress-induced sigma factor. An
hns
null mutation affects the cellular content of ς
S
in such a way that a remarkable accumulation of ς
S
is observed in the logarithmic growth phase, which results in enhanced expression of a number of ς
S
-dependent genes, including the
katE
gene. We isolated an extragenic mutation that affects the expression of the
katE-lacZ
fusion gene in the Δ
hns
background. The relevant gene was identified as
yhhP
, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of ς
S
. A deletion analysis of
yhhP
revealed that this small protein plays a fundamental role in the general physiology of
E. coli
. The
yhhP
-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.
Regulatory Conservation and Divergence of ς
32
Homologs from Gram-Negative Bacteria:
Serratia marcescens
,
Proteus mirabilis
,
Pseudomonas aeruginosa
, and
Agrobacterium tumefaciens
Authors: Nakahigashi Kenji , Yanagi Hideki , Yura Takashi
Published: December 2019

The heat shock response in
Escherichia coli
is mediated primarily by the
rpoH
gene, encoding ς
32
, which is specifically required for transcription of heat shock genes. A number of ς
32
homologs have recently been cloned from gram-negative bacteria that belong to the gamma or alpha subdivisions of the proteobacteria. We report here some of the regulatory features of several such homologs (RpoH) expressed in
E. coli
as well as in respective cognate bacteria. When expressed in an
E. coli
Δ
rpoH
strain lacking its own ς
32
, these homologs activated the transcription of heat shock genes (
groE
and
dnaK
) from the start sites normally used in
E. coli
. The level of RpoH in
Serratia marcescens
and
Pseudomonas aeruginosa
cells was very low at 30°C but was elevated markedly upon a shift to 42°C, as found previously with
E. coli
. The increased RpoH levels upon heat shock resulted from both increased synthesis and stabilization of the normally unstable RpoH protein. In contrast, the RpoH level in
Proteus mirabilis
was relatively high at 30°C and increased less markedly upon heat shock, mostly by increased synthesis; this ς
32
homolog was already stable at 30°C, and little further stabilization occurred upon the shift to 42°C. The increased synthesis of RpoH homologs in all these gamma proteobacteria was observed even in the presence of rifampin, suggesting that the induction occurred at the level of translation. Thus, the basic regulatory strategy of the heat shock response by enhancing the RpoH level is well conserved in the gamma proteobacteria, but some divergence in the actual mechanisms used occurred during evolution.
Mutations Affecting the α Subunit of
Bordetella pertussis
RNA Polymerase Suppress Growth Inhibition Conferred by Short C-Terminal Deletions of the Response Regulator BvgA
Authors: Stibitz Scott
Published: December 2019

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in
Bordetella pertussis
. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg
−
phenotype. When provided in
trans
, on the broad-host-range plasmid pRK290, under the control of the native
bvgAS
promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal
bvgAS
locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the α subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal
bvg
locus, three mutations which affected the α subunit of
B. pertussis
RNA polymerase were also isolated. Two of these resulted in increased levels of the α subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of
fha
was essentially normal, but expression of
ptx
was greatly reduced.
TheSaccharomyces cerevisiae YCC5(YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine
Authors: Schreve James L. , Sin Jennifer K. , Garrett Jinnie M.
Published: December 2019

The yeastYCC5gene encodes a putative amino acid permease and is homologous toGNP1(encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed thatAGP1expression is subject to nitrogen repression.
Identification and Analysis of “Extended −10” Promoters from Mycobacteria
Authors: Bashyam Murali D. , Tyagi Anil K.
Published: December 2019

Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the −10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their
Escherichia coli
counterparts. In contrast, the sequences in −35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their
E. coli
counterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847–4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the −10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a
Mycobacterium tuberculosis
promoter and a
Mycobacterium smegmatis
promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the −35 region. Comparative assessment of these extended −10 promoters in mycobacteria and
E. coli
suggests that functioning of the TGN motif in promoters of these two species is similar.
Coordinated Repression In Vitro of the Divergent
fepA-fes
Promoters of
Escherichia coli
by the Iron Uptake Regulation (Fur) Protein
Authors: Escolar Lucía , Pérez-Martín José , de Lorenzo Víctor
Published: December 2019

The mechanism involved in transcriptional repression of the
fepA-fes
divergent promoters of
Escherichia coli
by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped −35/−10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal −35/−10 hexamers was fully prevented by Fur-Mn
2+
bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the
fes
and
fepA
promoters could be displaced by Fur-Mn
2+
, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the
fes
promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the
fes
and
fepA
promoters and their coordinate regulation regardless of their different transcriptional activities. Comparisons with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.
Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase
Authors: Arens Jean Sippy , Hang Qi , Hwang Young , Tuma Bill , Max Sara , Feiss Mike
Published: December 2019

Terminase, an enzyme encoded by the
Nu1
and
A
genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions.
cosN
, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although
cosN
is rotationally symmetric, mutations in
cosN
have asymmetric effects. The
cosN
G
2
C mutation (a G-to-C change at position 2) in the left half of
cosN
reduces the phage yield 10-fold, whereas the symmetric mutation
cosN
C
11
G, in the right half of
cosN
, does not affect the burst size. The reduction in phage yield caused by
cosN
G
2
C is correlated with a defect in
cos
cleavage. Three suppressors of the
cosN
G
2
C mutation,

A-E
515
G

,

A-N
509
K

, and

A-R
504
C

, have been isolated that restore the yield of λ
cosN
G
2
C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ

A-E
515
G

,

A-N
509
K

, and

A-R
504
C

phages, which are
cosN
+
, also had wild-type burst sizes. In vitro
cos
cleavage experiments on
cosN
G
2
C C
11
G DNA showed that the rate of cleavage for

A-E
515
G

terminase is three- to fourfold higher than for wild-type terminase. The

A-E
515
G

mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ
cosN
G
2
C C
11
G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ
cosN
G
2
C C
11
G. In a λ
cosN
+
background, all amino acids tested at position 515 were functional. These results suggest that

A-E
515
G

plays an indirect role in extending the specificity of the endonuclease activity of λ terminase.
Lactobacillus casei
64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of
ptsH
and Different
gal
Mutants
Authors: Bettenbrock Katja , Siebers Ulrike , Ehrenreich Petra , Alpert Carl-Alfred
Published: December 2019

Galactose metabolism in
Lactobacillus casei
64H was analyzed by genetic and biochemical methods. Mutants with defects in
ptsH
,
galK
, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin.
ptsH
mutations abolish growth on lactose, cellobiose,
N
-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in
galK
mutants. A mutation in
galK
in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a
galK
mutation in combination with a mutation in
ptsH
completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.
Regulation of the
sol
Locus Genes for Butanol and Acetone Formation in
Clostridium acetobutylicum
ATCC 824 by a Putative Transcriptional Repressor
Authors: Nair Ramesh V. , Green Edward M. , Watson David E. , Bennett George N. , Papoutsakis Eleftherios T.
Published: December 2019

A gene (
orf1
, now designated
solR
) previously identified upstream of the aldehyde/alcohol dehydrogenase gene
aad
(R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871–885, 1994) was found to encode a repressor of the
sol
locus (
aad
,
ctfA
,
ctfB
and
adc
) genes for butanol and acetone formation in
Clostridium acetobutylicum
ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the
solR
start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the
sol
locus genes. Inactivation of
solR
in
C. acetobutylicum
via homologous recombination yielded mutants B and H (ATCC 824
solR
::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of
aad
, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.
Exopolysaccharide Biosynthesis in
Lactococcus lactis
NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone
Authors: van Kranenburg Richard , van Swam Iris I. , Marugg Joey D. , Kleerebezem Michiel , de Vos Willem M.
Published: December 2019

We used homologous and heterologous expression of the glycosyltransferase genes of the
Lactococcus lactis
NIZO B40
eps
gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.
Genetic and Biochemical Analyses of the
tec
Operon Suggest a Route for Evolution of Chlorobenzene Degradation Genes
Authors: Beil Stefan , Timmis Kenneth N. , Pieper Dietmar H.
Published: December 2019

The TecA broad-spectrum chlorobenzene dioxygenase of
Burkholderia
sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4,5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPβ incompatibility group. PCR analysis of the genetic environment of the
tec
genes indicated high similarity to the transposon-organized catabolic
tcb
chlorobenzene degradation genes of
Pseudomonas
sp. strain P51. Sequence analysis of the regions flanking the
tecA
genes revealed an upstream open reading frame (ORF) with high similarity to the
todF
2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of
Pseudomonas putida
F1 and a discontinuous downstream ORF showing high similarity to the
todE
catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing
meta
-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in
Escherichia coli
provided data consistent with the proposed relationships.
Evidence for a Chemiosmotic Model of Dehalorespiration in
Desulfomonile tiedjei
DCB-1
Authors: Louie Tai Man , Mohn William W.
Published: December 2019

Desulfomonile tiedjei
DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in
D. tiedjei
cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline
N
-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from
D. tiedjei
. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of
D. tiedjei
cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with
D. tiedjei
whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in
D. tiedjei
which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.
Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium,
Halomonas elongata
Authors: Ono Hisayo , Sawada Kazuhisa , Khunajakr Nonpanga , Tao Tao , Yamamoto Mihoko , Hiramoto Masayuki , Shinmyo Atsuhiko , Takano Mitsuo , Murooka Yoshikatsu
Published: December 2019

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in
Halomonas elongata
, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with
l
-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had

K
m

s of 9.1 mM for
l
-glutamate and 4.5 mM for
dl
-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-
N
-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a

K
m

of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.