bluuebunny/crossref_metadata_2025 huggingface dataset:
FtsI and FtsW Are Localized to the Septum in
Escherichia coli
Authors: Wang Lilin , Khattar Medhat K. , Donachie W. D. , Lutkenhaus Joe
Published: December 2019

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in
Escherichia coli
, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in
ftsZ
or
ftsA
mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.
Enhanced Secretory Production of a Single-Chain Antibody Fragment from
Bacillus subtilis
by Coproduction of Molecular Chaperones
Authors: Wu Sau-Ching , Ye Ruiqiong , Wu Xu-Chu , Ng Shi-Chung , Wong Sui-Lam
Published: December 2019

Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from
Bacillus subtilis
. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.
Distal Cleavage of 3-Chlorocatechol by an Extradiol Dioxygenase to 3-Chloro-2-Hydroxymuconic Semialdehyde
Authors: Riegert Ulrich , Heiss Gesche , Fischer Peter , Stolz Andreas
Published: December 2019

A 2,3-dihydroxybiphenyl 1,2-dioxygenase from the naphthalenesulfonate-degrading bacterium
Sphingomonas
sp. strain BN6 oxidized 3-chlorocatechol to a yellow product with a strongly pH-dependent absorption maximum at 378 nm. A titration curve suggested (de)protonation of an ionizable group with a pK
a
of 4.4. The product was isolated, purified, and converted, by treatment with diazomethane, to a dimethyl derivative and, by incubation with ammonium chloride, to a picolinic acid derivative. Mass spectra and
1
H and
13
C nuclear magnetic resonance (NMR) data for these two derivatives prove a 3-chloro-2-hydroxymuconic semialdehyde structure for the metabolite, resulting from distal (1,6) cleavage of 3-chlorocatechol. 3-Methylcatechol and 2,3-dihydroxybiphenyl are oxidized by this enzyme, in contrast, via proximal (2,3) cleavage.
Cascade Regulation of Dimethyl Sulfoxide Reductase (
dor
) Gene Expression in the Facultative Phototroph
Rhodobacter sphaeroides
2.4.1
T
Authors: Mouncey Nigel J. , Kaplan Samuel
Published: December 2019

Under anaerobic-dark growth conditions, in the presence of the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine
N
-oxide (TMAO),
Rhodobacter sphaeroides
2.4.1
T
respires anaerobically using the molybdoenzyme DMSO reductase (DMSOR). Genes encoding DMSOR and associated proteins are encoded by genes of the
dor
locus. Previously, we demonstrated that the expression of DMSOR is regulated by both the oxygen status of the cell via the FnrL protein and by the presence of DMSO or TMAO, presumably through the DorS-DorR two-component sensor-regulator system. Here we further investigate expression of the
dor
genes through the use of transcriptional
lacZ
fusions to the
dorS
,
dorR
, and
dorC
promoters. The expression of
dorC
::
lacZ
was strongly induced by the absence of oxygen and presence of DMSO. In accordance with our previous findings of DMSOR activity,
dorC
::
lacZ
expression was reduced by up to one-third when cells were grown photosynthetically in the presence of DMSO with medium or high light, compared to the expression observed after anaerobic-dark growth. The induction of
dorC
::
lacZ
expression in the presence of DMSO was dependent on the DorS and DorR proteins. Expression of the
dorS
and
dorR
genes was also induced in the absence of oxygen. In an FnrL mutant,
dorS
::
lacZ
expression was not induced when oxygen tensions in the media were lowered, in contrast to what occurred in the wild-type strain. The expression of
dorS
::
lacZ
and
dorR
::
lacZ
was dependent on the DorS and DorR proteins themselves, suggesting the importance of autoregulation. These results demonstrate a cascade regulation of
dor
gene expression, where the expression of the regulatory proteins DorS and DorR governs the downstream regulation of the
dorCBA
operon encoding the structural proteins of DMSOR.
A Spore Coat Protein, CotS, of
Bacillus subtilis
Is Synthesized under the Regulation of ς
K
and GerE during Development and Is Located in the Inner Coat Layer of Spores
Authors: Takamatsu Hiromu , Chikahiro Yukari , Kodama Takeko , Koide Hidekatsu , Kozuka Satoshi , Tochikubo Kunio , Watabe Kazuhito
Published: December 2019

The spore coat of
Bacillus subtilis
has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate
cotS
gene expression and CotS assembly into the coat layer of
B. subtilis
by Northern blot and Western blot analysis. Transcription of
cotS
mRNA was not detected in sporulating cells of ς
K
and
gerE
mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a
gerE
mutant or in the spores of mutants deficient in ς
E
, ς
F
, ς
G
, or ς
K
. CotS was detected in the sporangium but not in the spores of a
cotE
mutant. The sequence of the promoter region of
cotS
was similar to the consensus sequences for binding of ς
K
and GerE. These results demonstrate that ς
K
and GerE are required for
cotS
expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores.
Escherichia coli
Clone Sonnei (
Shigella sonnei
) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes
Authors: Lai Vincent , Wang Lei , Reeves Peter R.
Published: December 2019

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the
his
and
gnd
genes on the chromosome of
Escherichia coli
and related species.
Shigella sonnei
, essentially a clone of
E. coli
(
E. coli
clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of
E. coli
Sonnei among the 166 known
E. coli
serotype strains.
Cloning of the
Lactococcus lactis adhE
Gene, Encoding a Multifunctional Alcohol Dehydrogenase, by Complementation of a Fermentative Mutant of
Escherichia coli
Authors: Arnau José , Jørgensen Flemming , Madsen Søren M. , Vrang Astrid , Israelsen Hans
Published: December 2019

The
Lactococcus lactis adhE
gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an
L. lactis
genomic library in a fermentative mutant of
Escherichia coli
and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire
adhE
gene sequence. Analysis of
adhE
expression in
L. lactis
during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the
L. lactis
gene as a probe,
adhE
homologs were found in other industrially relevant lactic acid bacteria.
The
Caulobacter crescentus
Paracrystalline S-Layer Protein Is Secreted by an ABC Transporter (Type I) Secretion Apparatus
Authors: Awram Peter , Smit John
Published: December 2019

Caulobacter crescentus
is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3′ of
rsaA
, were found by screening a Tn
5
transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of
Pseudomonas aeruginosa
and the metalloprotease (PrtB) transport system of
Erwinia chrysanthemi
. The
prtB
and
aprA
genes were introduced into
C. crescentus
, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.
Preparation and Characterization of
Neisseria meningitidis
Mutants Deficient in Production of the Human Lactoferrin-Binding Proteins LbpA and LbpB
Authors: Bonnah Robert A. , Schryvers Anthony B.
Published: December 2019

Pathogenic members of the family
Neisseriaceae
produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens
Neisseria meningitidis
and
Moraxella catarrhalis
and the bovine pathogen
Moraxella bovis
by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285–297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the
N. meningitidis
B16B6
lbpA
gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with
N. meningitidis
isogenic mutants in which the
lbpA
gene and the ORF immediately upstream of
lbpA
(putative
lbpB
gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.
Altered Na
+
and Li
+
Homeostasis in
Saccharomyces cerevisiae
Cells Expressing the Bacterial Cation Antiporter NhaA
Authors: Ros Roc , Montesinos Consuelo , Rimon Abraham , Padan Etana , Serrano Ramón
Published: December 2019

The bacterial Na
+
(Li
+
)/H
+
antiporter NhaA has been expressed in the yeast
Saccharomyces cerevisiae
. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na
+
, Li
+
) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H
+
ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na
+
, Li
+
) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H
+
ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na
+
, Li
+
) ATPase and vacuolar H
+
ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na
+
, Li
+
) ATPase and vacuolar H
+
ATPase.
Transcriptional Regulation of
Alcaligenes eutrophus
Hydrogenase Genes
Authors: Schwartz Edward , Gerischer Ulrike , Friedrich Bärbel
Published: December 2019

Alcaligenes eutrophus
H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H
2
, supplying the organism with energy for autotrophic growth. The promoters of the structural genes for the SH and the MBH, P
SH
and P
MBH
, respectively, were identified by means of the primer extension technique. Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under conditions which repressed hydrogenase synthesis. The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Both promoters were dependent on the minor sigma factor ς
54
and on the hydrogenase regulator HoxA in vivo. P
SH
was stronger than P
MBH
under both heterotrophic and autotrophic growth conditions. The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures. The response regulator HoxA mediated low-level activation of P
SH
and P
MBH
in a heterologous system.
TheArcanobacterium(Actinomyces)pyogenesPlasmid pAP1 Is a Member of the pIJ101/pJV1 Family of Rolling Circle Replication Plasmids
Authors: Billington Stephen J. , Jost B. Helen , Songer J. Glenn
Published: December 2019

The 2.4-kb plasmid pAP1 fromArcanobacterium(Actinomyces)pyogeneshad sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate inEscherichia coliandCorynebacterium pseudotuberculosis, as well as inA. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.
The
Bacillus subtilis
AraE Protein Displays a Broad Substrate Specificity for Several Different Sugars
Authors: Krispin Oliver , Allmansberger Rudolf
Published: December 2019

Bacillus subtilis
168 is unable to grow on xylose and galactose as sole carbon sources, owing to the lack of specific transporters. We show that they are imported into the cell by the activity of AraE, an arabinose transporter whose synthesis is induced by
l
-arabinose.
The FixK2Protein Is Involved in Regulation of Symbiotic Hydrogenase Expression inBradyrhizobium japonicum
Authors: Durmowicz Meredith C. , Maier Robert J.
Published: December 2019

The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2in the symbiotic regulation of hydrogenase structural gene expression inBradyrhizobium japonicumhave been investigated. Bacteroids from FixJ and FixK2mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1mutant exhibit wild-type levels of hydrogenase activity. In β-galactosidase transcriptional assays with NifA and FixK2expression plasmids, the FixK2protein induces transcription from thehuppromoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2is involved in the transcriptional activation of symbiotic hydrogenase expression. By using β-galactosidase transcriptional fusion constructs containing successive truncations of thehuppromoter, the region of thehuppromoter required for regulation by FixK2was determined to be between 29 and 44 bp upstream of the transcription start site.
Activation of Bacteriophage Mu
mom
Transcription by C Protein Does Not Require Specific Interaction with the Carboxyl-Terminal Region of the α or ς
70
Subunit of
Escherichia coli
RNA Polymerase
Authors: Sun Weiyong , Hattman Stanley , Fujita Noboyuki , Ishihama Akira
Published: December 2019

Late in its growth cycle, transcription of the phage Mu
mom
promoter (P

mom

) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the α or ς
70
subunit of
Escherichia coli
RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both α and ς
70
.
Transcriptional Analysis of Essential Genes of the
Escherichia coli
Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous
Salmonella typhimurium
Gene Cluster
Authors: Zhang Yan , Cronan John E.
Published: December 2019

The genes encoding several key fatty acid biosynthetic enzymes (called the
fab
cluster) are clustered in the order
plsX-fabH-fabD-fabG-acpP-fabF
at min 24 of the
Escherichia coli
chromosome. A difficulty in analysis of the
fab
cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614–3620, 1996) is that several of these genes are essential for the growth of
E. coli
. We overcame this complication by use of the
fab
gene cluster of
Salmonella typhimurium
, a close relative of
E. coli
, to provide functions necessary for growth. The
S. typhimurium fab
cluster was isolated by complementation of an
E. coli fabD
mutant and was found to encode proteins with >94% homology to those of
E. coli
. However, the
S. typhimurium
sequences cannot recombine with the
E. coli
sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the
plsX
transcripts initiate at promoters located far upstream and include the upstream
rpmF
ribosomal protein gene, a promoter located upstream of the
plsX
coding sequence (probably within the upstream gene,
rpmF
) is sufficient for normal growth. We have also found that the
fabG
gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (Ω-Cm cassette) between the
fabD
and
fabG
genes of the
E. coli
chromosome abolished
fabG
transcription and blocked cell growth, thus providing the first indication that
fabG
is an essential gene. Insertion of the Ω-Cm cassette between
fabH
and
fabD
caused greatly decreased transcription of the
fabD
and
fabG
genes and slower cellular growth, indicating that
fabD
has only a weak promoter(s).
Induction of the SOS Response Increases the Efficiency of Global Nucleotide Excision Repair of Cyclobutane Pyrimidine Dimers, but Not 6-4 Photoproducts, in UV-Irradiated
Escherichia coli
Authors: Crowley David J. , Hanawalt Philip C.
Published: December 2019

Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair. In
Escherichia coli
, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response. We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin. Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively. These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage.
Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate
Authors: Montrone Marco , Eisenbach Michael , Oesterhelt Dieter , Marwan Wolfgang
Published: December 2019

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered
Escherichia coli
. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with
cheA
to
cheZ
deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.
Sed1p Is a Major Cell Wall Protein of
Saccharomyces cerevisiae
in the Stationary Phase and Is Involved in Lytic Enzyme Resistance
Authors: Shimoi Hitoshi , Kitagaki Hiroshi , Ohmori Hisanobu , Iimura Yuzuru , Ito Kiyoshi
Published: December 2019

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of
Saccharomyces cerevisiae
by incubation with
Rarobacter faecitabidus
protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the
SED1
gene.
SED1
was formerly identified as a multicopy suppressor of
erd2
, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of
SED1
had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.
The
fnr
Gene of
Bacillus licheniformis
and the Cysteine Ligands of the C-Terminal FeS Cluster
Authors: Klinger Anette , Schirawski Jan , Glaser Philippe , Unden Gottfried
Published: December 2019

In the facultatively anaerobic bacterium
Bacillus licheniformis
a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr from
Bacillus subtilis
, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from
B. subtilis
, this cluster (Cys226-X
2
-Cys229-X
4
-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the
B. licheniformis
gene to an
fnr
mutant of
B. subtilis
complemented the ability for synthesis of nitrate reductase during anaerobic growth.
Regulation of Biosynthesis of Individual Cellulases in
Thermomonospora fusca
Authors: Spiridonov Nikolay A. , Wilson David B.
Published: December 2019

Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium
Thermomonospora fusca
ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.
Roles of the Carboxy-Terminal Half of
Pseudomonas aeruginosa
Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association
Authors: Rawling Eileen G. , Brinkman Fiona S. L. , Hancock Robert E. W.
Published: December 2019

OprF, the major outer membrane protein of
Pseudomonas aeruginosa
, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on
P. aeruginosa
cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of
P. aeruginosa
in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.
Lack of
S
-Adenosylmethionine Results in a Cell Division Defect in
Escherichia coli
Authors: Newman E. B. , Budman L. I. , Chan E. C. , Greene R. C. , Lin R. T. , Woldringh C. L. , D’Ari R.
Published: December 2019

The enzyme
S
-adenosylmethionine (SAM) synthetase, the
Escherichia coli metK
gene product, produces SAM, the cell’s major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried
metK
gene prevents filamentation and restores normal growth to the
metK
mutant. This indicates that lack of SAM results in a division defect.
Low Ubiquinone Content in
Escherichia coli
Causes Thiol Hypersensitivity
Authors: Zeng H. , Snavely I. , Zamorano P. , Javor G. T.
Published: December 2019

Thiol hypersensitivity in a mutant of
Escherichia coli
(IS16) was reversed by complementation with a plasmid that carried the
ubiX
gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal
ubiX
genes indicated that both parent and mutant strains were
ubiX
mutants. The low ubiquinone content of IS16 was possibly caused by a
ubiD ubiX
genotype. A
ubiA
mutant also exhibited thiol hypersensitivity. Neither IS16 nor the
ubiA
mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb
−
phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery.
Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium
Pseudomonas putida
GM73
Authors: Kim Kwang , Lee Sungjin , Lee Kyunghee , Lim Dongbin
Published: December 2019

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium,
Pseudomonas putida
GM73, we carried out Tn
5
mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10
−6
). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn
5
-inserted genes, and the DNA sequence flanking Tn
5
was determined. From comparison with a sequence database, putative protein products encoded by
ttg
genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from
ttg8
did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.
Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product from
Bacillus subtilis
Authors: Inaoka Takashi , Matsumura Yoshinobu , Tsuchido Tetsuaki
Published: December 2019

Bacillus subtilis
was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated
sodA
, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of
sodA
. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant
Escherichia coli sodA sodB
, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of
B. subtilis sodA
.
Identification of a New Site for Ferrichrome Transport by Comparison of the FhuA Proteins of
Escherichia coli
,
Salmonella paratyphi
B,
Salmonella typhimurium
, and
Pantoea agglomerans
Authors: Killmann Helmut , Herrmann Christina , Wolff Helga , Braun Volkmar
Published: December 2019

The
fhuA
genes of
Salmonella paratyphi
B,
Salmonella typhimurium
, and
Pantoea agglomerans
were sequenced and compared with the known
fhuA
sequence of
Escherichia coli
. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in
E. coli
controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and φ80. All the FhuA proteins contained the region in the gating loops required in
E. coli
for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of
S. paratyphi
B which is infected by the
E. coli
phages, whereas two of the subdomains were deleted in
S. typhimurium
and
P. agglomerans
which are resistant to the
E. coli
phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated
E. coli
FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to φ80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of
S. paratyphi
B and
S. typhimurium
displayed
S. paratyphi
B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of
S. paratyphi
B and displayed
S. typhimurium
FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the
S. typhimurium
FhuA. The central segment of the
S. paratyphi
B FhuA flanked on both sides by
S. typhimurium
FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.
Control by Nutrients of Growth and Cell Cycle Progression in Budding Yeast, Analyzed by Double-Tag Flow Cytometry
Authors: Alberghina Lilia , Smeraldi Carla , Ranzi Bianca Maria , Porro Danilo
Published: December 2019

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual
Saccharomyces cerevisiae
cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing
S. cerevisiae
populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.
Isolation, Characterization, and Expression of the Gene Encoding the β Subunit of the Mitochondrial Processing Peptidase from
Blastocladiella emersonii
Authors: Rocha Cíntia Renata Costa , Gomes Suely Lopes
Published: December 2019

A 2.3-kb
Bam
HI-
Kpn
I fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the β subunit of the
Blastocladiella
mitochondrial processing peptidase (β-MPP). The predicted β-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that β-MPP mRNA levels decrease significantly during
B. emersonii
sporulation, reaching basal levels in the zoospore stage. The amount of β-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.
The Succinyl and Acetyl Modifications of Succinoglycan Influence Susceptibility of Succinoglycan to Cleavage by the
Rhizobium meliloti
Glycanases ExoK and ExsH
Authors: York Gregory M. , Walker Graham C.
Published: December 2019

In
Rhizobium meliloti
(
Sinorhizobium meliloti
) cultures, the endo-1,3-1,4-β-glycanases ExoK and ExsH depolymerize nascent high-molecular-weight (HMW) succinoglycan to yield low-molecular-weight (LMW) succinoglycan. We report here that the succinyl and acetyl modifications of succinoglycan influence the susceptibility of succinoglycan to cleavage by these glycanases. It was previously shown that
exoH
mutants, which are blocked in the succinylation of succinoglycan, exhibit a defect in the production of LMW succinoglycan. We have determined that
exoZ
mutants, which are blocked in the acetylation of succinoglycan, exhibit an increase in production of LMW succinoglycan. For both wild-type and
exoZ
mutant strains, production of LMW succinoglycan is dependent on the
exoK
+
and
exsH
+
genes, implying that the ExoK and ExsH glycanases cleave HMW succinoglycan to yield LMW succinoglycan. By supplementing cultures of glycanase-deficient strains with exogenously added ExoK or ExsH, we have demonstrated directly that the absence of the acetyl group increases the susceptibility of succinoglycan to cleavage by ExoK and ExsH, that the absence of the succinyl group decreases the susceptibility of succinoglycan to cleavage, and that the succinyl effect outweighs the acetyl effect for succinoglycan lacking both modifications. Strikingly, nonsuccinylated succinoglycan actually can be cleaved by ExoK and ExsH to yield LMW succinoglycan, but only when the glycanases are added to cultures at greater than physiologically relevant concentrations. Thus, we conclude that the molecular weight distribution of succinoglycan in
R. meliloti
cultures is determined by both the levels of ExoK and ExsH glycanase expression and the susceptibility of succinoglycan to cleavage.
Oligohistidine Tag Mutagenesis of the λ Holin Gene
Authors: Smith David L. , Young Ry
Published: December 2019

Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. Recently, the holin
S
gene of phage λ was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D. L. Smith, D. K. Struck, J. M. Scholtz, and R. Young, J. Bacteriol. 180:2531–2540, 1998). Numerous locations within the
S
gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function. The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized. A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein. Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function. The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required.
One of Two OsmC Homologs in
Bacillus subtilis
Is Part of the ς
B
-Dependent General Stress Regulon
Authors: Völker Uwe , Andersen Kasper Krogh , Antelmann Haike , Devine Kevin M. , Hecker Michael
Published: December 2019

In this report we present the identification and analysis of two
Bacillus subtilis
genes,
yklA
and
ykzA
, which are homologous to the partially RpoS-controlled
osmC
gene from
Escherichia coli
. The
yklA
gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter. Expression of
ykzA
is not medium dependent but increases dramatically when cells are exposed to stress and starvation. This stress-induced increase in
ykzA
expression is absolutely dependent on the alternative sigma factor ς
B
, which controls a large stationary-phase and stress regulon.
ykzA
is therefore another example of a gene common to the RpoS and ς
B
stress regulons of
E. coli
and
B. subtilis
, respectively. The composite complex expression pattern of the two
B. subtilis
genes is very similar to the expression profile of
osmC
in
E. coli.
Phosphate Assimilation in
Rhizobium
(
Sinorhizobium
)
meliloti
: Identification of a
pit
-Like Gene
Authors: Bardin Sylvie D. , Voegele Ralf T. , Finan Turlough M.
Published: December 2019

Rhizobium meliloti
mutants defective in the
phoCDET
-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix
−
). We have previously reported that two classes of second-site mutations can suppress the Fix
−
phenotype of
phoCDET
mutants to Fix
+
. Here we show that one of these suppressor loci (
sfx1
) contains two genes,
orfA
and
pit
, which appear to form an operon transcribed in the order
orfA-pit
. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the
orfA
transcription start site. This mutation increased the level of
orfA-pit
transcription. These data, together with previous biochemical evidence, show that the
orfA-pit
genes encode a P
i
transport system that is expressed in wild-type cells grown with excess P
i
but repressed in cells under conditions of P
i
limitation. In
phoCDET
mutant cells,
orfA-pit
expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases
orfA-pit
expression compensating for the deficiencies in phosphate assimilation and symbiosis of the
phoCDET
mutants.
Regulation of
hepA
of
Anabaena
sp. Strain PCC 7120 by Elements 5′ from the Gene and by
hepK
Authors: Zhu Jinsong , Kong Renqiu , Wolk C. Peter
Published: December 2019

In
Anabaena
spp., synthesis of the heterocyst envelope polysaccharide, required if the cell is to fix dinitrogen under aerobic conditions, is dependent on the gene
hepA
. A transcriptional start site of
hepA
was localized 104 bp 5′ from its translational initiation codon. A 765-bp open reading frame, denoted
hepC
, was found farther upstream. Inactivation of
hepC
led to constitutive expression of
hepA
and prevented the synthesis of heterocyst envelope polysaccharide. However, the glycolipid layer of the heterocyst envelope was synthesized. A
hepK
mutation blocked both the synthesis of the heterocyst envelope polysaccharide and induction of
hepA
. The predicted product of
hepK
resembles a sensory protein-histidine kinase of a two-component regulatory system. Analysis of the region between
hepC
and
hepA
indicated that DNA sequences required for the induction of
hepA
upon nitrogen deprivation are present between bp −574 and −440 and between bp −340 and −169 relative to the transcriptional start site of
hepA
. Gel mobility shift assays provided evidence that one or more proteins bind specifically to the latter sequence. The Fox box sequence downstream from
hepA
appeared inessential for the induction of
hepA
.
Analysis of the
puc
Operon Promoter from
Rhodobacter capsulatus
Authors: Nickens David G. , Bauer Carl E.
Published: December 2019

Expression of the
Rhodobacter capsulatus puc
operon, which codes for structural polypeptides of the light-harvesting-II peripheral antenna complex, is highly regulated in response to alterations in oxygen tension and light intensity. To obtain an understanding of the
puc
promoter region we report the high-resolution 5′ mapping of the
puc
mRNA transcriptional start site and DNA sequence analysis of the
puc
upstream regulatory sequence (
puc
URS). A ς
70
-type promoter sequence was identified (
puc
P1) which has a high degree of sequence similarity with carotenoid and bacteriochlorophyll biosynthesis promoters. Inspection of the DNA sequence also indicated the presence of two CrtJ and four integration host factor (IHF) binding sites. Transcriptional fusions of the
puc
URS fused to
lacZ
also confirmed that
puc
promoter activity is regulated by the transcriptional regulators IHF, CrtJ, and RegA. Gel retardation analysis using cell extracts indicates that mutations in IHF and RegA disrupt protein binding to DNA fragments containing the
puc
URS.
Agrobacterium
VirE2 Proteins Can Form a Complex with T Strands in the Plant Cytoplasm
Authors: Gelvin Stanton B.
Published: December 2019

Wild-type VirE2 and VirD2 proteins from
Agrobacterium tumefaciens
contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An
A. tumefaciens virE2 virD2
ΔNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.