bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppression of the
ptsH
Mutation in
Escherichia coli
and
Salmonella typhimurium
by a DNA Fragment from
Lactobacillus casei
Authors: Monedero Vicente , Postma Pieter W. , Pérez-Martínez Gaspar 
Published: December 2019

ABSTRACT

A DNA fragment from
Lactobacillus casei
that restores growth to
Escherichia coli
and
Salmonella typhimurium ptsH
mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of
ptsH
homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the
ptsH
phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.
Chemotaxis in
Borrelia burgdorferi
Authors: Shi Wenyuan , Yang Zhaomin , Geng Yongzhi , Wolinsky Lawrence E. , Lovett Michael A. 
Published: December 2019

ABSTRACT

Borrelia burgdorferi
is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of
B. burgdorferi
required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of
B. burgdorferi
. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of
B. burgdorferi
. Rabbit serum was found to be an attractant for
B. burgdorferi
, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g.,
Spirochaeta aurantia
),
B. burgdorferi
did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of
B. burgdorferi.
Direct Sulfhydrylation for Methionine Biosynthesis in
Leptospira meyeri
Authors: Belfaiza J. , Martel A. , Margarita D. , Saint Girons I. 
Published: December 2019

ABSTRACT

A gene library of the
Leptospira meyeri
serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a
Leptospira
DNA fragment carrying
metY
complemented
Escherichia coli
strains carrying mutations in
metB
. The latter gene encodes cystathionine γ-synthase, an enzyme which catalyzes the second step of the methionine biosynthetic pathway. The
metY
gene is 1,304 bp long and encodes a 443-amino-acid protein with a molecular mass of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the
Leptospira metY
product has a high degree of similarity to those of
O
-acetylhomoserine sulfhydrylases from
Aspergillus nidulans
and
Saccharomyces cerevisiae
. A lower degree of sequence similarity was also found with bacterial cystathionine γ-synthase. The
L. meyeri metY
gene was overexpressed under the control of the T7 promoter. MetY exhibits an
O
-acetylhomoserine sulfhydrylase activity. Genetic, enzymatic, and physiological studies reveal that the transsulfuration pathway via cystathionine does not exist in
L. meyeri
, in contrast to the situation found for fungi and some bacteria. Our results indicate, therefore, that the
L. meyeri
MetY enzyme is able to perform direct sulfhydrylation for methionine biosynthesis by using
O
-acetylhomoserine as a substrate.
Reverse Gyrase from the Hyperthermophilic Bacterium
Thermotoga maritima
: Properties and Gene Structure
Authors: de la Tour Claire Bouthier , Portemer Christiane , Kaltoum Habib , Duguet Michel 
Published: December 2019

ABSTRACT

The hyperthermophilic bacterium
Thermotoga maritima
MSB8 possesses a reverse gyrase whose enzymatic properties are very similar to those of archaeal reverse gyrases. It catalyzes the positive supercoiling of the DNA in an Mg
2+
- and ATP-dependent process. Its optimal temperature of activity is around 90°C, and it is highly thermostable. We have cloned and DNA sequenced the corresponding gene (
T. maritima topR
). This is the first report describing the analysis of a gene encoding a reverse gyrase in bacteria. The
T. maritima topR
gene codes for a protein of 1,104 amino acids with a deduced molecular weight of 128,259, a value in agreement with that estimated from the denaturing gel electrophoresis of the purified enzyme. Like its archaeal homologs, the
T. maritima
reverse gyrase exhibits helicase and topoisomerase domains, and its sequence matches very well the consensus sequence for six reverse gyrases now available. Phylogenetic analysis shows that all reverse gyrases, including the
T. maritima
enzyme, form a very homogeneous group, distinct from the type I 5′ topoisomerases of the TopA subfamily, for which we have previously isolated a representative gene in
T. maritima
(
topA
). The coexistence of these two distinct genes, coding for a reverse gyrase and an ω-like topoisomerase, respectively, together with the recent description of a gyrase in
T. maritima
(O. Guipaud, E. Marguet, K. M. Noll, C. Bouthier de la Tour, and P. Forterre, Proc. Natl. Acad. Sci. USA 94:10606–10611, 1977) addresses the question of the control of the supercoiling in this organism.
Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of
Pseudomonas putida
GJ31
Authors: Kaschabek Stefan R. , Kasberg Thomas , Müller Dagmar , Mars Astrid E. , Janssen Dick B. , Reineke Walter 
Published: December 2019

ABSTRACT

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of
Pseudomonas putida
GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native
M
r
value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.
YopD of
Yersinia pestis
Plays a Role in Negative Regulation of the Low-Calcium Response in Addition to Its Role in Translocation of Yops
Authors: Williams Andrew W. , Straley Susan C. 
Published: December 2019

ABSTRACT

Yersinia pestis
produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37°C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a
yopD
mutation in both wild-type and secretion-defective (
ysc
)
Y. pestis
to determine if the
lcrH
phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single
yopD
mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the
yopD
mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.
Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring
Authors: Hugenholtz Philip , Pitulle Christian , Hershberger Karen L. , Pace Norman R. 
Published: December 2019

ABSTRACT

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609–1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or
Bacteria
-specific rDNA primers and cloned. Unique rDNA types among >300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (≥98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing
Calderobacterium
and the sulfate reducers
Thermodesulfovibrio
and
Thermodesulfobacterium
, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing
Aquificales
and the sulfate-reducing δ-
Proteobacteria
. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that
Archaea
dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.
Staphylococcus aureus cap5O
and
cap5P
Genes Functionally Complement Mutations Affecting Enterobacterial Common-Antigen Biosynthesis in
Escherichia coli
Authors: Kiser Kevin B. , Lee Jean C. 
Published: December 2019

ABSTRACT

The
Staphylococcus aureus cap5P
and
cap5O
genes of the type 5 capsule biosynthetic locus restore enterobacterial common-antigen expression to
Escherichia coli
mutants defective in
rffE
and
rffD
gene expression, respectively. Cap5P and Cap5O likely function as UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase enzymes, respectively, in the synthesis of the capsule precursor UDP-ManNAcA.
The ATP Synthase
atpHAGDC
(F
1
) Operon from
Rhodobacter capsulatus
Authors: Borghese Roberto , Crimi Massimo , Fava Luca , Melandri Bruno Andrea 
Published: December 2019

ABSTRACT

The
atpHAGDC
operon of
Rhodobacter capsulatus
, containing the five genes coding for the F
1
sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying
atp
deletions in the
R. capsulatus
chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.
Conversion of NfsA, the Major
Escherichia coli
Nitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in
Vibrio harveyi
, by a Single Amino Acid Substitution
Authors: Zenno Shuhei , Kobori Toshiro , Tanokura Masaru , Saigo Kaoru 
Published: December 2019

ABSTRACT

NfsA is the major oxygen-insensitive nitroreductase of
Escherichia coli
, similar in amino acid sequence to Frp, a flavin reductase of
Vibrio harveyi
. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.
Domain Structure of the ATP-Binding-Cassette Protein MalK of
Salmonella typhimurium
as Assessed by Coexpressed Half Molecules and LacK′-′MalK Chimeras
Authors: Schmees Günter , Schneider Erwin 
Published: December 2019

ABSTRACT

ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of
Salmonella typhimurium
and
Escherichia coli
is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of
S. typhimurium
. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a
malK
strain and displayed substantial regulatory activity. The same regulatory activity was obtained when
malKC1
was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.
Gram-Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria
Authors: Li Zusheng , Clarke Anthony J. , Beveridge Terry J. 
Published: December 2019

ABSTRACT

Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (
Citrobacter
,
Enterobacter
,
Escherichia
,
Klebsiella
,
Morganella
,
Proteus
,
Salmonella
, and
Shigella
strains), lysed many gram-positive (including
Mycobacterium
) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected.
Pseudomonas aeruginosa
PAO1 vesicles possessed the most lytic activity.
The Nine Genes of the
Nocardia lactamdurans
Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region
Authors: Enguita Francisco J. , Coque Juan Jose R. , Liras Paloma , Martin Juan F. 
Published: December 2019

ABSTRACT

The nine biosynthesis genes of the
Nocardia lactamdurans
cephamycin cluster are expressed as three different mRNAs initiating at promoters
latp
,
cefDp
, and
pcbABp
, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (
latp
and
cefDp
) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (
Streptomyces
antibiotic regulatory proteins) family. The
lat
gene is transcribed in a single monocistronic transcript initiating at
latp
. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (
pcbAB
,
pcbC
,
cmcI
,
cmcJ
,
cefF
, and
cmcH
) started at
pcbABp
. A third polycistronic mRNA corresponding to the
cefD
and
cefE
genes started at
cefDp
.
Identification and Recombinant Expression of a
Mycobacterium avium
Rhamnosyltransferase Gene (
rtfA
) Involved in Glycopeptidolipid Biosynthesis
Authors: Eckstein Torsten M. , Silbaq Fauzi S. , Chatterjee Delphi , Kelly Nathan J. , Brennan Patrick J. , Belisle John T. 
Published: December 2019

ABSTRACT

The
Mycobacterium avium
complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the
M. avium
complex. We previously described the
ser2
gene cluster, encoding the synthesis of the haptenic oligosaccharide (2,3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (
ser2A
) encoding a putative rhamnosyltransferase. Sequencing of the
ser2A
locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in
Mycobacterium smegmatis
, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed
rtfA
, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of
rtfA
will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple
M. avium
complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.
Endochitinase Is Transported to the Extracellular Milieu by theeps-Encoded General Secretory Pathway ofVibrio cholerae
Authors: Connell Terry D. , Metzger Daniel J. , Lynch Jennifer , Folster Jason P. 
Published: December 2019

ABSTRACTThechiAgene ofVibrio choleraeencodes a polypeptide which degrades chitin, a homopolymer ofN-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans.chiAhas a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of thechiAopen reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity byV. choleraerequiredchiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for β,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes ofV. choleraeand by the closely related bacteriumAeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by theepssystem. Theepssystem is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential foreps-dependent secretion.
Multiple In Vivo Roles for the −12-Region Elements of Sigma 54 Promoters
Authors: Wang Lei , Gralla Jay D. 
Published: December 2019

ABSTRACT
Alignment of sigma 54-dependent promoters indicates conservation of two sequence elements. Six nucleotides in the downstream −12 element were mutated individually to each nonconsensus nucleotide. mRNA levels were measured in vivo for each promoter under strongly activating conditions. The results showed that the consensus sequence was not the strongest promoter. Instead, the −12 consensus element consists of two subregions that behave differently when mutated. Single changes in the upstream TTT consensus subregion can lead to increases in transcription, whereas single changes in the downstream GC(A/T) can lead to decreases in transcription. Selected double mutations with changes in both subregions were constructed and studied in vivo. No double mutation increased promoter strength, and some decreased it. Mutant promoters were also assayed under nonactivating conditions in vivo. No mRNA was detected in 23 of the 24 promoters tested. However, one double mutant showed substantial levels of transcript, indicating that the −12 sequence was capable of specifying basal transcription under nonactivating conditions. Overall, the results show that the −12 region has multiple roles in transcription in vivo, including modulating both basal and induced RNA levels.