bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism
Authors: Nemecek-Marshall Michele , Wojciechowski Cheryl , Wagner William P. , Fall Ray 
Published: December 2019

ABSTRACT

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the
Vibrionaceae
. Sixteen
Vibrio
isolates, two
Listonella
species, and two
Photobacterium angustum
isolates produced acetone in the presence of
l
-leucine.
Shewanella
isolates produced much less acetone. Growth of
Vibrio splendidus
and
P. angustum
in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium.
l
-Leucine, but not
d
-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of
l
-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only α-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d
7
)-
l
-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.
Differential and Independent Roles of a ς
32
Homolog (RpoH) and an HrcA Repressor in the Heat Shock Response of
Agrobacterium tumefaciens
Authors: Nakahigashi Kenji , Ron Eliora Z. , Yanagi Hideki , Yura Takashi 
Published: December 2019

ABSTRACT

The heat shock response in alpha proteobacteria is unique in that a combination of two regulators is involved: a positive regulator, RpoH (ς
32
homolog), found in the alpha, beta, and gamma proteobacteria, and a negative regulator, HrcA, widely distributed in eubacteria but not in the gamma proteobacteria. To assess the differential roles of the two regulators in these bacteria, we cloned the
hrcA-grpE
operon of
Agrobacterium tumefaciens
, analyzed its transcription, and constructed deletion mutants lacking RpoH and/or HrcA. The
ΔrpoH
mutant and
ΔrpoH ΔhrcA
double mutant were unable to grow above 30°C. Whereas the synthesis of heat shock proteins (e.g., DnaK, GroEL, and ClpB) was transiently induced upon temperature upshift from 25 to 37°C in the wild type, such induction was not observed in the
ΔrpoH
mutant, except that GroEL synthesis was still partially induced. By contrast, the
ΔhrcA
mutant grew normally and exhibited essentially normal heat induction except for a higher level of GroEL expression, especially before heat shock. The
ΔrpoH ΔhrcA
double mutant showed the combined phenotypes of each of the single mutants. The amounts of
dnaK
and
groE
transcripts before and after heat shock, as determined by primer extension, were consistent with those of the proteins synthesized. The cellular level of RpoH but not HrcA increased significantly upon heat shock. We conclude that RpoH plays a major and global role in the induction of most heat shock proteins, whereas HrcA plays a restricted role in repressing
groE
expression under nonstress conditions.
Gradual Alterations in Cell Wall Structure and Metabolism in Vancomycin-Resistant Mutants of
Staphylococcus aureus
Authors: Sieradzki Krzysztof , Tomasz Alexander 
Published: December 2019

ABSTRACT

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant
Staphylococcus aureus
strain COL (MIC of methicillin, 800 μg/ml; MIC of vancomycin, 1.5 μg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to β-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.
Regulation of Expression of the
adhE
Gene, Encoding Ethanol Oxidoreductase in
Escherichia coli
: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS
Authors: Membrillo-Hernández Jorge , Lin E. C. C. 
Published: December 2019

ABSTRACT

The
adhE
gene of
Escherichia coli
, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of
adhE
is dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the
lacZ
gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activates
adhE
transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.
RNase E Enzymes from
Rhodobacter capsulatus
and
Escherichia coli
Differ in Context- and Sequence-Dependent In Vivo Cleavage within the Polycistronic
puf
mRNA
Authors: Heck Claudia , Evguenieva-Hackenberg Elena , Balzer Angelika , Klug Gabriele 
Published: December 2019

ABSTRACT

The 5′
pufQ
mRNA segment and the
pufLMX
mRNA segment of
Rhodobacter capsulatus
exhibit different stabilities. Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage. While
Rhodobacter
RNase E does not discriminate between the different sequences present around the cleavage sites within
pufQ
and
pufL
,
Escherichia coli
RNase E shows preference for the sequence harboring more A and U residues.
Archaeal Nucleosome Positioning by CTG Repeats
Authors: Sandman Kathleen , Reeve John N. 
Published: December 2019

ABSTRACT

DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)
6
and (CTG)
8
repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)
4
-, (CTG)
5
-, or (CTG)
3
AA(CTG)
3
-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.
Membrane-Mimicking Entities Induce Structuring of the Two-Peptide Bacteriocins Plantaricin E/F and Plantaricin J/K
Authors: Hauge Håvard Hildeng , Mantzilas Dimitris , Eijsink Vincent G. H. , Nissen-Meyer Jon 
Published: December 2019

ABSTRACT

Lactobacillus plantarum
C11 produces plantaricin E/F (PlnE/F) and plantaricin J/K (PlnJ/K), two bacteriocins whose activity depends on the complementary action of two peptides (PlnE and PlnF; PlnJ and PlnK). Three of the individual Pln peptides possess some antimicrobial activity, but the highest bacteriocin activity is obtained by combining complementary peptides in about a one-to-one ratio. Circular dichroism was used to study the structure of the Pln peptides under various conditions. All four peptides were unstructured under aqueous conditions but adopted a partly alpha-helical structure in the presence of trifluoroethanol, micelles of dodecylphosphocholine, and negatively charged dioleoylphosphoglycerol (DOPG) liposomes. Far less structure was induced by zwitterionic dioleoylglycerophosphocholine liposomes, indicating that a net negative charge on the phospholipid bilayer is important for a structure-inducing interaction between (positively charged) Pln peptides and a membrane. The structuring of complementary peptides was considerably enhanced when both (PlnE and PlnF or PlnJ and PlnK) were added simultaneously to DOPG liposomes. Such additional structuring was not observed in experiments with trifluoroethanol or dodecylphosphocholine, indicating that the apparent structure-inducing interaction between complementary Pln peptides requires the presence of a phospholipid bilayer. The amino acid sequences of the Pln peptides are such that the alpha-helical structures adopted upon interaction with the membrane and each other are amphiphilic in nature, thus enabling membrane interactions.
Assembly of the K40 Antigen in
Escherichia coli
: Identification of a Novel Enzyme Responsible for Addition of
l
-Serine Residues to the Glycan Backbone and Its Requirement for K40 Polymerization
Authors: Amor Paul A. , Yethon Jeremy A. , Monteiro Mario A. , Whitfield Chris 
Published: December 2019

ABSTRACT

Escherichia coli
O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40
LPS
backbone structure is composed of a trisaccharide repeating unit of
N
-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an
l
-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145–161, 1997). Here, we demonstrate that insertional inactivation of
orf1
results in the accumulation of a semirough (SR)-K40
LPS
form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40
LPS
reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that
orf1
encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of
orf3
also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the
l
-serine substitution of GlcA. These results show that (i)
orf3
encodes the enzyme responsible for the addition of the
l
-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with
l
-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.
A Four-Dimensional View of Assembly of a Morphogenetic Protein during Sporulation in
Bacillus subtilis
Authors: Price Kirsten D. , Losick Richard 
Published: December 2019

ABSTRACT

We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium
Bacillus subtilis
. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium
Clostridium acetobutylicum
, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like
spoIVA
, are under the control of the mother cell transcription factor ς
E
. One such gene was
spoVM
, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.
Coordinate Intracellular Expression of
Salmonella
Genes Induced during Infection
Authors: Heithoff Douglas M. , Conner Christopher P. , Hentschel Ute , Govantes Fernando , Hanna Philip C. , Mahan Michael J. 
Published: December 2019

ABSTRACT

Salmonella typhimurium
in vivo-induced (
ivi
) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg
2+
, Fe
2+
, and PhoPQ. All of the seven
ivi
fusions that are induced by both low pH and low Mg
2+
(e.g.,
iviVI-A
) are activated by the PhoPQ regulatory system. Iron-responsive
ivi
fusions include those induced under iron limitation (e.g.,
entF
) as well as one induced by iron excess but only in the absence of PhoP (
pdu
). Intracellular expression studies showed that each of the pH- and Mg
2+
-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407. Each
ivi
fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed
ivi
genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types. Investigation of
ivi
expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments.
Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in
Salmonella typhimurium
: Incorporation of Stable Isotope-Labeled Glycine and Formate
Authors: Enos-Berlage Jodi L. , Downs Diana M. 
Published: December 2019

ABSTRACT

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in
Salmonella typhimurium
. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-
13
C]glycine and [
13
C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a
purF
mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.
Poly-3-Hydroxybutyrate Degradation in
Rhizobium (Sinorhizobium) meliloti
: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase
Authors: Aneja P. , Charles T. C. 
Published: December 2019

ABSTRACT

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (
bdhA
) from
Rhizobium (Sinorhizobium) meliloti
. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The
R. meliloti
Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily.
bdhA
is the first gene transcribed in an operon that also includes
xdhA
, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς
54
consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a
rpoN
mutant of
R. meliloti
was able to utilize 3-hydroxybutyrate as a sole carbon source. The
R. meliloti bdhA
gene is able to confer upon
Escherichia coli
the ability to utilize 3-hydroxybutyrate as a sole carbon source. An
R. meliloti bdhA
mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a
lacZ
transcriptional fusion to
bdhA
demonstrated that gene expression is growth phase associated.
Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium
Authors: Adams David G. , Ashworth Denise , Nelmes Brenda 
Published: December 2019

ABSTRACTThe cell walls of a number of filamentous, gliding cyanobacteria of the genusOscillatoriawere examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.
Diversity of
radA
Genes from Cultured and Uncultured
Archaea
: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker
Authors: Sandler Steven J. , Hugenholtz Philip , Schleper Christa , DeLong Edward F. , Pace Norman R. , Clark Alvin J. 
Published: December 2019

ABSTRACT

Archaea
-specific
radA
primers were used with PCR to amplify fragments of
radA
genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15
radA
genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of
Crenarcheota
. The two most deeply branching archaeal
radA
genes found had some amino acid deletion and insertion patterns characteristic of bacterial
recA
genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.
Gene Duplication and Multiplicity of Collagenases in
Clostridium histolyticum
Authors: Matsushita Osamu , Jung Chang-Min , Katayama Seiichi , Minami Junzaburo , Takahashi Yukie , Okabe Akinobu 
Published: December 2019

ABSTRACT

Clostridium histolyticum
collagenase contains a number of different active components. Previously we have shown that
colH
encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (
colG
). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that
colG
and
colH
encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome.
C. histolyticum colG
is more similar to
C. perfringens colA
than to
colH
in terms of domain structure. Both
colG
and
colA
have a homologous gene,
mscL
, at their 3′ ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to
C. histolyticum
and
C. perfringens
and that further divergence of the parent gene produced
colG
and
colA
.