bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Alkyl Hydroperoxide Reductase (
ahpCF
) Gene in Oxidative Stress Defense of the Obligate Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Smith C. Jeffrey 
Published: December 2019

ABSTRACT

In this study we report the identification and role of the alkyl hydroperoxide reductase (
ahp
) gene in
Bacteroides fragilis
. The two components of
ahp
,
ahpC
, and
ahpF
, are organized in an operon, and the deduced amino acid sequences revealed that
B. fragilis
AhpCF shares approximately 60% identity to orthologues in other gram-positive and gram-negative bacteria. Northern blot hybridization analysis of total RNA showed that the
ahpCF
genes were transcribed as a polycistronic 2.4-kb mRNA and that
ahpC
also was present as a 0.6-kb monocistronic mRNA.
ahpC
and
ahpCF
mRNAs were induced approximately 60-fold following H
2
O
2
treatment or oxygen exposure of the parent strain but were constitutive in a peroxide-resistant strain. Further investigation using an
ahpCF
′::β-xylosidase gene transcriptional fusion confirmed that
ahpCF
had lost normal regulation in the peroxide-resistant strain compared to the parent. The
ahpCF
mutant was more sensitive to growth inhibition and mutagenesis by organic peroxides than the parent strain, as determined by disk inhibition assays and the frequency of mutation to fusidic acid resistance. This finding suggests that the
ahp
genes play an important role in peroxide resistance in
B. fragilis
. Under anaerobic conditions, we observed increases in the number of spontaneous fusidic acid-resistant mutants of five- and sevenfold in
ahpCF
and
ahpF
strain backgrounds, respectively, and eightfold in the
ahpCF katB
double mutant strain compared to the parent and
katB
strains. In addition,
ahpCF
,
ahpF
, and
ahpCF katB
mutants were slightly more sensitive to oxygen exposure than the parent strain. Moreover, the isolation of a strain with enhanced aerotolerance and high-level resistance to alkyl hydroperoxides from an
ahpCF katB
parent suggests that the physiological responses to peroxide toxicity and to the toxic effects of molecular oxygen are overlapping and complex in this obligate anaerobe.
The
vsp
Locus of
Mycoplasma bovis
: Gene Organization and Structural Features
Authors: Lysnyansky Inessa , Sachse Konrad , Rosenbusch Ricardo , Levisohn Sharon , Yogev David 
Published: December 2019

ABSTRACT

Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen
Mycoplasma bovis
were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the
M. bovis
chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copy
vsp
genes organized in a chromosomal cluster were identified and characterized. All
vsp
genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each
vsp
coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various
vsp
genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each
vsp
gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the
vsp
genes and within the
vsp
coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the
vsp
locus of
M. bovis
.
Characterization ofEnterococcus faecalisAlkaline Phosphatase and Use in IdentifyingStreptococcus agalactiaeSecreted Proteins
Authors: Lee Martin H. , Nittayajarn Aphakorn , Ross R. Paul , Rothschild Cynthia B. , Parsonage Derek , Claiborne Al , Rubens Craig E. 
Published: December 2019

ABSTRACTWe have identified and characterized anEnterococcus faecalisalkaline phosphatase (AP, encoded byphoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases fromBacillus subtilis. Expression ofphoZinEscherichia coli,E. faecalis,Streptococcus agalactiae(group B streptococcus [GBS]), orStreptococcus pyogenes(group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme’s subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). InE. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant,phoZΔss, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, andE. faecalisand is expected to be useful in a variety of gram-positive bacteria.
Essential Role of the Iron-Regulated Outer Membrane Receptor FauA in Alcaligin Siderophore-Mediated Iron Uptake in
Bordetella
Species
Authors: Brickman Timothy J. , Armstrong Sandra K. 
Published: December 2019

ABSTRACT

Phenotypic analysis using heterologous host systems localized putative
Bordetella pertussis
ferric alcaligin transport genes and Fur-binding sequences to a 3.8-kb genetic region downstream from the
alcR
regulator gene. Nucleotide sequencing identified a TonB-dependent receptor family homolog gene,
fauA
, predicted to encode a polypeptide with high amino acid sequence similarity with known bacterial ferric siderophore receptors. In
Escherichia coli
, the
fauA
genes of both
B. pertussis
and
Bordetella bronchiseptica
directed the production of a 79-kDa polypeptide, approximating the predicted size of the mature FauA protein.
B. bronchiseptica fauA
insertion mutant BRM17 was unable to utilize ferric alcaligin, and in complementation analyses ferric alcaligin utilization was restored to this mutant by supplying the wild-type
fauA
gene in
trans
. Mutant BRM18, carrying a nonpolar in-frame
fauA
deletion mutation, was defective in ferric alcaligin utilization and
55
Fe-ferric alcaligin uptake and no longer produced a 79-kDa iron-regulated outer membrane protein. In complementation analyses, BRM18 merodiploids bearing the wild-type
fauA
gene in
trans
regained ferric alcaligin siderophore transport and utilization functions and produced the 79-kDa protein. Analysis of a plasmid-borne
fauA-lacZ
operon fusion confirmed that
fauA
is subject to iron regulation at the transcriptional level and that
cis
-acting transcriptional control elements mediating
fauA
iron repressibility reside within the 3.8-kb
Pst
I
fauA
DNA region. Moreover, expression of the
fauA-lacZ
fusion gene under iron starvation conditions was shown to be
alcR
dependent. FauA is a 79-kDa iron-regulated outer membrane receptor protein required for transport and utilization of ferric alcaligin siderophore complexes by
Bordetella
species.
Abiotic Surface Sensing and Biofilm-Dependent Regulation of Gene Expression in
Escherichia coli
Authors: Prigent-Combaret Claire , Vidal Olivier , Dorel Corinne , Lejeune Philippe 
Published: December 2019

ABSTRACT

To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in
Escherichia coli
K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless
lacZ
gene, was performed with an
ompR234
adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the
proU
operon), the colanic acid exopolysaccharide (
wca
locus, formerly called
cps
), tripeptidase T (
pepT
), and the nickel high-affinity transport system (
nikA
). On the other hand, the syntheses of flagellin (
fliC
) and of a putative protein of 92 amino acids (
f92
) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.
A Mutant HemA Protein with Positive Charge Close to the N Terminus Is Stabilized against Heme-Regulated Proteolysis in
Salmonella typhimurium
Authors: Wang Liying , Wilson Sandra , Elliott Thomas 
Published: December 2019

ABSTRACT
The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.
rsmC
of the Soft-Rotting Bacterium
Erwinia carotovora
subsp.
carotovora
Negatively Controls Extracellular Enzyme and Harpin
Ecc
Production and Virulence by Modulating Levels of Regulatory RNA (
rsmB
) and RNA-Binding Protein (RsmA)
Authors: Cui Yaya , Mukherjee Asita , Dumenyo C. Korsi , Liu Yang , Chatterjee Arun K. 
Published: December 2019

ABSTRACT

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin
Ecc
(the elicitor of hypersensitive reaction) in
Erwinia carotovora
subsp.
carotovora
is regulated by RsmA, an RNA-binding protein, and
rsmB
, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219–234, 1998). We have cloned and characterized a novel regulatory gene,
rsmC
, that activates RsmA production and represses extracellular enzyme and harpin
Ecc
production,
rsmB
transcription, and virulence in
E. carotovora
subsp.
carotovora
. In an
rsmC
knockout mutant of
E. carotovora
subsp.
carotovora
Ecc71 carrying the chromosomal copy of the wild-type
rsmA
+
allele, the basal levels of Pel, Peh, Cel, Prt, and harpin
Ecc
as well as the amounts of
rsmB
,
pel-1
,
peh-1
,
celV
, and

hrpN
Ecc

transcripts are high, whereas the levels of
rsmA
transcripts and RsmA protein are low. Furthermore, the expression of an
rsmA-lacZ
gene fusion is lower in the RsmC
−
mutant than in the RsmC
+
parent. Conversely, the expression of an
rsmB-lacZ
operon fusion is higher in the RsmC
−
mutant than in the RsmC
+
parent. These observations establish that RsmC negatively regulates
rsmB
transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC
−
mutant, an RsmA
−
mutant, and an RsmA
−
RsmC
−
double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on
rsmA
and
rsmB
. Exoprotein production by the RsmC
−
mutant is partially dependent on the quorum sensing signal,
N
-(3-oxohexanoyl)-
l
-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of
rsmC
sequences in
E. carotovora
subsp.
atroseptica
,
E. carotovora
subsp.
betavasculorum
, and
E. carotovora
subsp.
carotovora
. These findings collectively support the idea that
rsmA
and
rsmB
expression in these plant pathogenic
Erwinia
species is controlled by RsmC or a functional homolog of RsmC.
Sequence Analysis of the Gene Encoding Amylosucrase from
Neisseria polysaccharea
and Characterization of the Recombinant Enzyme
Authors: Potocki De Montalk G. , Remaud-Simeon M. , Willemot R. M. , Planchot V. , Monsan P. 
Published: December 2019

ABSTRACT

The
Neisseria polysaccharea
gene encoding amylosucrase was subcloned and expressed in
Escherichia coli
. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)
8
-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione
S
-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.
Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination
Authors: Peña Carol E. A. , Kahlenberg J. Michelle , Hatfull Graham F. 
Published: December 2019

ABSTRACT

The temperate mycobacteriophage L5 integrates site specifically into the genomes of
Mycobacterium smegmatis
,
Mycobacterium tuberculosis
, and
Mycobacterium bovis
bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5
attP
site and the mycobacterial
attB
site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that
attP
, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of
attB
capture and formation of products. The arm-type integrase binding sites within
attP
play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the
attP
core, while an additional pair of sites, P1-P2, is required for interaction with
attB
.
Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases
Authors: Bzymek Malgorzata , Saveson Catherine J. , Feschenko Vladimir V. , Lovett Susan T. 
Published: December 2019

ABSTRACT

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In
Escherichia coli
, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.
Temporal Expression of the
Bacillus subtilis secA
Gene, Encoding a Central Component of the Preprotein Translocase
Authors: Herbort Markus , Klein Michael , Manting Erik H. , Driessen Arnold J. M. , Freudl Roland 
Published: December 2019

ABSTRACT

In
Bacillus subtilis
, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the
secA
gene, encoding a central component of the
B. subtilis
preprotein translocase. We found that
secA
and the downstream gene (
prfB
) constitute an operon that is transcribed from a vegetative (ς
A
-dependent) promoter located upstream of
secA
. Furthermore, using different independent methods, we found that
secA
expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of
secA
sharply decreased to a low basal level. Since at the time of maximal
secA
transcription the secretion activity of
B. subtilis
strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the
B. subtilis
protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed.
Localization of FtsI (PBP3) to the Septal Ring Requires Its Membrane Anchor, the Z Ring, FtsA, FtsQ, and FtsL
Authors: Weiss David S. , Chen Joseph C. , Ghigo Jean-Marc , Boyd Dana , Beckwith Jon 
Published: December 2019

ABSTRACT

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of
Escherichia coli
, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of
ftsI
at the normal chromosomal locus and a genetic fusion of
ftsI
to the green fluorescent protein (
gfp
) at the lambda attachment site.
gfp-ftsI
was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of β-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.
Motility of
Helicobacter pylori
Is Coordinately Regulated by the Transcriptional Activator FlgR, an NtrC Homolog
Authors: Spohn Gunther , Scarlato Vincenzo 
Published: December 2019

ABSTRACT

ς
54
is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of
Helicobacter pylori
genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified ς
54
-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the ς
28
-regulated
flaA
flagellin gene promoter, while changes in DNA topology repress transcription of the ς
54
-regulated
flaB
flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in
H. pylori
shows similarities with that in enterobacteriaceae and
Caulobacter
.
Role of Glycosylation at Ser63 in Production of Soluble Pilin in Pathogenic
Neisseria
Authors: Marceau Michael , Nassif Xavier 
Published: December 2019

ABSTRACT

Pilus-mediated adhesion is essential in the pathogenesis of
Neisseria meningitidis
(MC) and
Neisseria gonorrhoeae
(GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.
Identification of SycN, YscX, and YscY, Three New Elements of the
Yersinia
Yop Virulon
Authors: Iriarte Maite , Cornelis Guy R. 
Published: December 2019

ABSTRACT

The Yop virulon allows
Yersinia
spp. to resist the immune response of the host by injecting harmful proteins into host cells. We identified three new elements of the Yop virulon: SycN, required for normal secretion of YopN, and YscX and YscY, two new components of the secretion machinery.