bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Evidence that
S
-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases
Authors: Val Dale L. , Cronan John E. 
Published: December 2019

ABSTRACT

Many gram-negative bacteria synthesize
N
-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In
Escherichia coli
, synthesis of
N
-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a
fadD
mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the
fad
regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular
S
-adenosylmethionine caused by expression of bacteriophage T3
S
-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from
S
-adenosylmethionine.
Negative Regulation of IS
2
Transposition by the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Authors: Hu Shiau-Ting , Wang Hsuan-Chen , Lei Guang-Sheng , Wang Shao-Hung 
Published: December 2019

ABSTRACT

Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS
2
promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS
2
transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS
2
promoter. A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above. With an IS
2
promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS
2
promoter. IS
2
was found to transpose at a frequency approximately 200-fold higher in an
Escherichia coli
host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS
2
transposition.
Isolation and Characterization of Three
Streptococcus pneumoniae
Transformation-Specific Loci by Use of a
lacZ
Reporter Insertion Vector
Authors: Pestova Ekaterina V. , Morrison Donald A. 
Published: December 2019

ABSTRACT

Although more than a dozen new proteins are produced when
Streptococcus pneumoniae
cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random
lacZ
transcriptional fusion library was constructed in
S. pneumoniae
by using the insertional
lacZ
reporter vector pEVP3. Screening the library for clones with competence-specific β-galactosidase (β-Gal) production yielded three insertion mutants with induced β-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the
lacZ
reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of
Bacillus subtilis com
operons. One locus contained open reading frames (ORFs) homologous to the
comEA
and
comEC
genes in
B. subtilis
but lacked a
comEB
homolog. A second locus contained four ORFs with homology to the
B. subtilis comG
gene ORFs 1 to 4, but
comG
gene ORFs 5 to 7 were replaced in
S. pneumoniae
with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an
erm
cassette for gene disruptions.
Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of
Agrobacterium tumefaciens
Authors: Lai Erh-Min , Kado Clarence I. 
Published: December 2019

ABSTRACT

Previous studies have implicated the obligatory requirement for the
vir
regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from
Agrobacterium tumefaciens
to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the
virB
operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in
Escherichia coli
. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced
A. tumefaciens
had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production of
vir
gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires other
virB
genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of
A. tumefaciens.
Joint Transcriptional Control ofxpsR, the Unusual Signal Integrator of theRalstonia solanacearumVirulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator
Authors: Huang Jianzhong , Yindeeyoungyeon Wandee , Garg Ram P. , Denny Timothy P. , Schell Mark A. 
Published: December 2019

ABSTRACTRalstonia(Pseudomonas)solanacearumis a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of theepsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsRpromoter (PxpsR) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theepspromoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of PxpsRto show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of PxpsR; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of PxpsRrequires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate PxpsR. (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect PxpsRactivation or regulation.
Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily
Authors: Zhang Xiaolan , Zhu Liuqin , Deutscher Murray P. 
Published: December 2019

ABSTRACT

Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of
Escherichia coli
. The purified protein is an α2 dimer of 40 kDa. NH
2
-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as
yjeR
(
o204a
), a previously reported open reading frame located at 94 min on the
E. coli
chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of
yjeR
led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose that
yjeR
be renamed
orn
. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.
Functional Similarity between Archaeal and Bacterial CorA Magnesium Transporters
Authors: Smith Ronald L. , Gottlieb Erik , Kucharski Lisa M. , Maguire Michael E. 
Published: December 2019

ABSTRACT

The constitutively expressed CorA Mg
2+
transporter is the major Mg
2+
influx system of
Salmonella typhimurium
and
Escherichia coli
. Genomic sequence data indicated the presence of a homolog in the archaeal organism
Methanococcus jannaschii
. The putative
M. jannaschii
CorA was expressed in an Mg
2+
-transport-deficient strain of
S. typhimurium
to determine its functional characteristics. The archaeal CorA homolog is a functional Mg
2+
uptake system when expressed in
S. typhimurium
and has properties which are highly similar to those of the normal CorA transporter of
S. typhimurium
despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal. This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.