bluuebunny/crossref_metadata_2025_split huggingface dataset:
The “Pro” Sequence of the Sporulation-Specific ς Transcription Factor ς
E
Directs It to the Mother Cell Side of the Sporulation Septum
Authors: Ju Jingliang , Haldenwang W. G. 
Published: December 2019

ABSTRACT

ς
E
, a mother cell-specific transcription factor of sporulating
Bacillus subtilis
, is derived from an inactive precursor protein (pro-ς
E
). Activation of ς
E
occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-ς
E
amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-ς
E
and green fluorescent protein (GFP) to visualize the intracellular location of pro-ς
E
by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-ς
E
::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-ς
E
::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-ς
E
::GFP, and presumably pro-ς
E
, to the mother cell is likely to be the reason why ς
E
activity is restricted to that compartment.
Resistance to Bacitracin as Modulated by an
Escherichia coli
Homologue of the Bacitracin ABC Transporter BcrC Subunit from
Bacillus licheniformis
Authors: Harel Yael Meller , Bailone Adriana , Bibi Eitan 
Published: December 2019

ABSTRACT

A small open reading frame from the
Escherichia coli
chromosome,
bcrC
EC
, encodes a homologue to the BcrC subunit of the bacitracin permease from
Bacillus licheniformis
. We show that disruption of the chromosomal
bcrC
EC
gene causes bacitracin sensitivity and, conversely, that BcrC
EC
confers bacitracin resistance when expressed from a multicopy plasmid.
Chromosome Segregation and Cell Division Defects in
recBC sbcBC ruvC
Mutants of
Escherichia coli
Authors: Zahradka Davor , Vlahović Ksenija , Petranović Mirjana , Petranović Drago 
Published: December 2019

ABSTRACT

The RuvC protein is important for DNA recombination and repair in
Escherichia coli
. The present work shows that a
ruvC
null mutation introduced into a
recBC sbcBC
background causes severe defects in chromosome segregation and cell division. Both defects were found to result from abortive recombination initiated by the RecA protein.
Functional Expression of hMYH, a Human Homolog of the
Escherichia coli
MutY Protein
Authors: Slupska Malgorzata M. , Luther Wendy M. , Chiang Ju-Huei , Yang Hanjing , Miller Jeffrey H. 
Published: December 2019

ABSTRACT

We have previously described the
hMYH
cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885–3892, 1996). Here, we report that the enzyme expressed from an
hMYH
cDNA clone in
Escherichia coli
complements the mutator phenotype in a
mutY
mutant and can remove A from an A · 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A · G mismatch in vitro.
recD sbcB sbcD
Mutants Are Deficient in Recombinational Repair of UV Lesions by RecBC
Authors: Seigneur Marie , Ehrlich S. Dusko , Michel Bénédicte 
Published: December 2019

ABSTRACT

In
recD sbcB sbcD
mutants, repair of UV-irradiated DNA is strongly RecF dependent, indicating that RecBC is inactive. This finding suggests that exonuclease V, exonuclease I (SbcB), and the SbcCD nuclease play a redundant role in vivo, which is essential for the recombination activity of the RecBC complex during UV repair.
Molecular Characterization ofeutFMutants ofSalmonella typhimuriumLT2 IdentifieseutFLesions as Partial-Loss-of-FunctiontonBAlleles
Authors: Thomas Michael G. , O’Toole George A. , Escalante-Semerena Jorge C. 
Published: December 2019

ABSTRACTTheeutFlocus ofSalmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization ofeutFmutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining theeutFlocus, Δ903andeutF1115, are partial-loss-of-functiontonBalleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonBgene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carryingeutFalleles. Molecular analysis of the Δ903allele identified a deletion that resulted in the fusion of the 3′ end oftonBwith the 3′ end oftrpA. In-frame translation of thetonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903showed that the new mutation was a deletion of two nucleotides at thetonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine andeutoperon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, theeutFlocus does not exist.
Cell Wall Teichoic Acid Glycosylation in
Listeria monocytogenes
Serotype 4b Requires
gtcA
, a Novel, Serogroup-Specific Gene
Authors: Promadej N. , Fiedler F. , Cossart P. , Dramsi S. , Kathariou S. 
Published: December 2019

ABSTRACT

We have identified a novel gene,
gtcA
, involved in the decoration of cell wall teichoic acid of
Listeria monocytogenes
serotype 4b with galactose and glucose. Insertional inactivation of
gtcA
brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on teichoic acid. Interestingly, the composition of membrane-associated lipoteichoic acid was not affected. Complementation of the mutants with the cloned
gtcA
in
trans
restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within
L. monocytogenes
, sequences homologous to
gtcA
were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b,
gtcA
appears to be the first member of a bicistronic operon which includes a gene with homology to
Bacillus subtilis rpmE
, encoding ribosomal protein L31. In contrast to
gtcA
, the latter gene appears conserved among all screened serotypes of
L. monocytogenes
.
Regulation of Hexuronate Utilization in
Bacillus subtilis
Authors: Mekjian Kathleen R. , Bryan Edward M. , Beall Bernard W. , Moran Charles P. 
Published: December 2019

ABSTRACT

We have identified a locus essential for galacturonate utilization in
Bacillus subtilis
. Genes homologous to
Escherichia coli
and
Erwinia chrysanthemi
glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the
B. subtilis
chromosome. A mutant of
B. subtilis
containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a ς
A
-dependent promoter,
exu
P1, located upstream from the first ORF. The eighth ORF in this cluster (the
exu
locus) encodes a LacI and GalR homolog that negatively regulated expression from
exu
P1. A 26-bp inverted repeat sequence centered 15 bp downstream from the
exu
P1 start point of transcription acted in
cis
to negatively regulate expression from
exu
P1 under noninducing conditions. Expression from the
exu
P1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A ς
E
-dependent promoter,
exu
P2, was localized between the second and third ORFs and was active during sporulation.
Septal Localization of FtsQ, an Essential Cell Division Protein in
Escherichia coli
Authors: Chen Joseph C. , Weiss David S. , Ghigo Jean-Marc , Beckwith Jon 
Published: December 2019

ABSTRACT

Septation in
Escherichia coli
requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The
gfp-ftsQ
gene complements a null mutation in
ftsQ
. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.
The
phn
Genes of
Burkholderia
sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism
Authors: Laurie Andrew D. , Lloyd-Jones Gareth 
Published: December 2019

ABSTRACT

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical
nah
-like (
nah
,
ndo
,
pah
, and
dox
) sequences. Here we report the description of a divergent set of PAH catabolic genes, the
phn
genes, which although isofunctional to the classical
nah
-like genes, show very low homology. This
phn
locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb
Hin
dIII fragment from phenanthrene-degrading
Burkholderia
sp. strain RP007. The
phn
genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in
Escherichia coli
the recombinant
phn
enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein α and β subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the
phn
catabolic genes are two putative regulatory genes,
phnR
and
phnS
. Sequence homology suggests that
phnS
is a LysR-type transcriptional activator and that
phnR
, which is divergently transcribed with respect to
phnSFECDAcAdB
, is a member of the ς
54
-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS.
Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in
Bacillus megaterium
Authors: McCool Gabriel J. , Cannon Maura C. 
Published: December 2019

ABSTRACT

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in
Bacillus megaterium
11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5
pha
genes,
phaP
, -
Q
, -
R
, -
B
, and -
C
. The
phaP
and -
Q
genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream,
phaR
, -
B
, and -
C
were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to
Escherichia coli
and to a PhaC
−
mutant of
Pseudomonas putida
gave a Pha
+
phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.
S-Methylmethionine Metabolism inEscherichia coli
Authors: Thanbichler Martin , Neuhierl Bernhard , Böck August 
Published: December 2019

ABSTRACTSelenium-accumulatingAstragalusspp. contain an enzyme which specifically transfers a methyl group fromS-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed thatEscherichia colipossesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is anS-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants inyagDwhich also possess defects inmetEandmetHare unable to utilizeS-methylmethionine for growth, whereas ametE metHdouble mutant still grows onS-methylmethionine. Upstream ofyagDand overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in ametE metHgenetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter forS-methylmethionine. Methionine but notS-methylmethionine in the medium reduces the amount ofyagDprotein. This and the existence of four MET box motifs upstream ofyfkDindicate that the two genes are members of the methionine regulon. The physiological roles of theykfDandyagDproducts appear to reside in the acquisition ofS-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.
Escherichia coli
Lrp (Leucine-Responsive Regulatory Protein) Does Not Directly Regulate Expression of the
leu
Operon Promoter
Authors: Landgraf Jeffrey R. , Boxer Jonathan A. , Calvo Joseph M. 
Published: December 2019

ABSTRACT

Studies by R. Lin et al. (J. Bacteriol. 174:1948–1955, 1992) suggested that the
Escherichia coli leu
operon might be a member of the Lrp regulon. Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in
leu
operon expression between
lrp
+
and
lrp
strains. Furthermore, when
leuP-lacZ
transcriptional fusions that lacked the
leu
attenuator were used, expression from the
leu
promoter varied less than twofold between
lrp
+
and
lrp
strains, irrespective of whether or not excess leucine was added to the medium. The simplest explanation of the observations of Lin et al. is that the known elevated leucine transport capacity of
lrp
strains (S. A. Haney et al., J. Bacteriol. 174:108–115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of
leu
operon expression.
Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in
Staphylococcus aureus
Infection
Authors: Ji Yinduo , Marra Andrea , Rosenberg Martin , Woodnutt Gary 
Published: December 2019

ABSTRACT

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted a
tet
regulatory expression system for use in
Staphylococcus aureus
, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the
S. aureus
alpha-toxin gene (
hla
) in antisense orientation downstream of the
tet
promoter system and introduced the construct into
S. aureus
. Induced antisense
hla
RNA downregulated chromosomally derived
hla
gene expression in vitro approximately 14-fold. Similarly, induction of
hla
antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of
S. aureus
infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the
tet
regulatory system functions efficiently in
S. aureus
and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.
The
Oenococcus oeni clpX
Homologue Is a Heat Shock Gene Preferentially Expressed in Exponential Growth Phase
Authors: Jobin Michel-Philippe , Garmyn Dominique , Diviès Charles , Guzzo Jean 
Published: December 2019

ABSTRACT

Using degenerated primers from conserved regions of previously studied
clpX
gene products, we cloned the
clpX
gene of the malolactic bacterium
Oenococcus oeni
. The
clpX
gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed
clpX
gene products from other organisms. An open reading frame located upstream of the
clpX
gene was identified as the
tig
gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic
clpX
and
tig
mRNAs and also showed an increase in
clpX
mRNA amount after a temperature shift from 30 to 42°C. The
clpX
transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike
hsp18
, the gene encoding one of the major small heat shock proteins of
Oenococcus oeni
,
clpX
expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5′ end of
clpX
mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as
Escherichia coli.
Activation of the Cryptic
aac(6′)-Iy
Aminoglycoside Resistance Gene of
Salmonella
by a Chromosomal Deletion Generating a Transcriptional Fusion
Authors: Magnet Sophie , Courvalin Patrice , Lambert Thierry 
Published: December 2019

ABSTRACT

Salmonella enterica
subsp.
enterica
serotype Enteritidis BM4361 and BM4362 were isolated from the same patient. BM4361 was susceptible to aminoglycosides, whereas BM4362 was resistant to tobramycin owing to synthesis of a 6′-
N
-acetyltransferase type I [AAC(6′)-I]. Comparative analysis of nucleotide sequences, pulsed-field gel electrophoresis patterns, and Southern hybridizations indicated that the chromosomal
aac(6′)-Iy
genes for the enzyme in both strains were identical and that BM4362 derived from BM4361 following a ca. 60-kb deletion that occurred 1.5 kb upstream from the resistance gene. Northern hybridizations showed that
aac(6′)-Iy
was silent in BM4361 and highly expressed in BM4362 due to a transcriptional fusion. Primer extension mapping identified the transcriptional start site for
aac(6′)-Iy
in BM4362: 5 bp downstream from the promoter of the
nmpC
gene. Study of the distribution of
aac(6′)-Iy
by PCR and Southern hybridization with a specific probe indicated that the gene, although not found in
S. enterica
subsp.
arizonae
, was specific for
Salmonella
. In this bacterial genus,
aac(6′)-Iy
was located downstream from a cluster of seven open reading frames analogous to an
Escherichia coli
locus that encodes enzymes putatively involved in carbohydrate transport or metabolism. This genomic environment suggests a role in the catabolism of a specific sugar for AAC(6′)-Iy in
Salmonella
.
Acetate Metabolism in a
pta
Mutant of
Escherichia coli
W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival
Authors: Chang Dong-Eun , Shin Sooan , Rhee Joon-Shick , Pan Jae-Gu 
Published: December 2019

ABSTRACT

In order to study the physiological role of acetate metabolism in
Escherichia coli
, the growth characteristics of an
E. coli
W3100
pta
mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The
pta
mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,
d
-lactate, and
l
-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the
pta
mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The
pta
mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the
pta
mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (
phbCAB
) from
Alcaligenes eutrophus
, indicating that the growth defect of the
pta
mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that
E. coli
excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the
pta
mutant.
Stable Packaging of Phage PRD1 DNA Requires Adsorption Protein P2, Which Binds to the IncP Plasmid-Encoded Conjugative Transfer Complex
Authors: Grahn A. Marika , Caldentey Javier , Bamford Jaana K. H. , Bamford Dennis H. 
Published: December 2019

ABSTRACT

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent

K
d

of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.