bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine
Authors: Burke Stephen A. , Lo Sam L. , Krzycki Joseph A. 
Published: December 2019

ABSTRACT

Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in
Methanosarcina barkeri
MS. The gene encoding MMCP (
mtmC
) is located directly upstream of the gene encoding MMAMT (
mtmB
). The gene encoding MT2-A (
mtbA
) was found 1.1 kb upstream of
mtmC
, but no obvious open reading frame was found in the intergenic region between
mtbA
and
mtmC
. A single monocistronic transcript was found for
mtbA
that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried
mtmCB
. The larger transcript also encoded
mtmP
, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of
mtmC
. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of
mtmB
was interrupted by a single in-frame, midframe, UAG codon which was also found in
mtmB
from
M. barkeri
NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of
mtmB
in this methanogen.
Characterization of the Earliest Known
Staphylococcus aureus
Plasmid Encoding a Multidrug Efflux System
Authors: Paulsen Ian T. , Brown Melissa H. , Skurray Ronald A. 
Published: December 2019

ABSTRACT

The staphylococcal
qacB
-encoding multidrug resistance plasmid pSK156, isolated from a clinical strain dating from 1951, was characterized. Comparison of the regions flanking
qacB
with other
qacA
- and
qacB
-encoding plasmids provided insights into the evolution and dissemination of these multidrug efflux genes and led to the detection of the earliest known copy of the insertion sequence IS
257
.
Characterization of the Maleylacetate Reductase MacA of
Rhodococcus opacus
1CP and Evidence for the Presence of an Isofunctional Enzyme
Authors: Seibert Volker , Kourbatova Elena M. , Golovleva Ludmila A. , Schlömann Michael 
Published: December 2019

ABSTRACT

Maleylacetate reductases (EC
1.3.1.32
) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene
macA
, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain
Rhodococcus opacus
1CP. Sequencing of
macA
showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned
Rhodococcus
maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of
macA
, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with
macA
encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the
R. opacus
maleylacetate reductase gene
macA
clearly is not part of a specialized chlorocatechol gene cluster.
The
apeE
Gene of
Salmonella typhimurium
Encodes an Outer Membrane Esterase Not Present in
Escherichia coli
Authors: Carinato Maria E. , Collin-Osdoby Patricia , Yang Xioming , Knox Tina M. , Conlin Christopher A. , Miller Charles G. 
Published: December 2019

ABSTRACT

Salmonella typhimurium apeR
mutations lead to overproduction of an outer membrane-associated
N
-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the
apeE
gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the
S. typhimurium apeE
gene as well as some properties of the esterase that it encodes. The predicted product of
apeE
is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium
Photorhabdus luminescens
. The
Salmonella
esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C
6
to C
16
fatty acid
p
-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing
Salmonella
spp. from related organisms makes use of the ability of
Salmonella
to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the
apeE
gene product is the enzyme in
Salmonella
uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that
Escherichia coli
K-12 does not contain a close analog of
apeE
, and it appears that the
apeE
gene is contained in a region of DNA present in
Salmonella
but not in
E. coli
.
Dual Promoters Are Responsible for Transcription Initiation of the
fla/che
Operon in
Bacillus subtilis
Authors: Estacio W. , Anna-Arriola S. Santa , Adedipe M. , Márquez-Magaña L. M. 
Published: December 2019

ABSTRACT

The
fla/che
region contains more than 30 genes required for flagellar synthesis and chemotaxis in
Bacillus subtilis
, including the gene for the flagellum-specific ς
D
factor,
sigD
. Sequence and primer extension data demonstrate that a P
A
promoter immediately upstream of
flgB
, henceforth referred to as the
fla/che
P
A
, and the P
D-3
promoter are active in vivo. Transcription from the P
D-3
element is dependent on ς
D
activity and is regulated by the flagellum-specific negative regulator, FlgM. In a strain containing a deletion of
fla/che
P
A
(P
A
Δ), ς
D
protein was not detected, demonstrating that the
fla/che
P
A
is necessary for wild-type expression of the
sigD
gene. Thus,
sigD
is part of the >26-kb
fla/che
operon. Consistent with a lack of detectable ς
D
protein, the P
A
Δ strain grows as long filaments and does not express a ς
D
-dependent
hag
::
lacZ
reporter construct. These phenotypes are indicative of a lack of
sigD
expression or complete inhibition of ς
D
activity by FlgM. However, ς
D
activity is found in a double mutant containing the P
A
Δ and a null mutation in
flgM
. The double mutant no longer grows as long filaments, and expression of
hag
::
lacZ
is partially restored. These data demonstrate that a low level of ς
D
activity does exist in the P
A
Δ mutant but can be detected only in the presence of a null mutation in
flgM
. Therefore, normal expression of
sigD
may also involve another promoter(s) within the
fla/che
operon.
Spo0A Mutants of
Bacillus subtilis
with Sigma Factor-Specific Defects in Transcription Activation
Authors: Hatt Janet K. , Youngman Philip 
Published: December 2019

ABSTRACT

The transcription factor Spo0A of
Bacillus subtilis
has the unique ability to activate transcription from promoters that require different forms of RNA polymerase holoenzyme. One class of Spo0A-activated promoter, which includes
spoIIEp
, is recognized by RNA polymerase associated with the primary sigma factor, sigma A (ς
A
); the second, which includes
spoIIAp
, is recognized by RNA polymerase associated with an early-sporulation sigma factor, sigma H (ς
H
). Evidence suggests that Spo0A probably interacts directly with RNA polymerase to activate transcription from these promoters. To identify residues of Spo0A that may be involved in transcriptional activation, we used PCR mutagenesis of the entire
spo0A
gene and designed a screen using two distinguishable reporter fusions,
spoIIE-gus
and
spoIIA-lacZ
. Here we report the identification and characterization of five mutants of Spo0A that are specifically defective in activation of ς
A
-dependent promoters while maintaining activation of ς
H
-dependent promoters. These five mutants identify a 14-amino-acid segment of Spo0A, from residue 227 to residue 240, that is required for transcriptional activation of ς
A
-dependent promoters. This region may define a surface or domain of Spo0A that makes direct contacts with ς
A
-associated holoenzyme.
A Minisatellite Sequence within the Propeptide Region of the Vacuolar Carboxypeptidase Y Gene of
Schizosaccharomyces pombe
Authors: Ingavale Susham S. , Kaur Rupinder , Aggarwal Parul , Bachhawat Anand K. 
Published: December 2019

ABSTRACT

We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast
Schizosaccharomyces pombe
. The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene. The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of
S. pombe
(
S. pombe
var.
pombe
and
S. pombe
var.
malidevorans
). The minisatellite sequence, however, appeared to be species specific and was absent in other members of the
Schizosaccharomyces
genus. This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.
General Stress Transcription Factor ς
B
and Sporulation Transcription Factor ς
H
Each Contribute to Survival of
Bacillus subtilis
under Extreme Growth Conditions
Authors: Gaidenko Tatiana A. , Price Chester W. 
Published: December 2019

ABSTRACT

The general stress response of the bacterium
Bacillus subtilis
is controlled by the ς
B
transcription factor. Here we show that loss of ς
B
reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol. We further show that loss of the developmental transcription factor ς
H
also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.
Biochemical Properties and Substrate Specificities of a Recombinantly Produced
Azotobacter vinelandii
Alginate Lyase
Authors: Ertesvåg Helga , Erlien Frode , Skjåk-Bræk Gudmund , Rehm Bernd H. A. , Valla Svein 
Published: December 2019

ABSTRACT

Alginate is a polysaccharide composed of β-
d
-mannuronic acid (M) and α-
l
-guluronic acid (G). An
Azotobacter vinelandii
alginate lyase gene,
algL
, was cloned, sequenced, and expressed in
Escherichia coli
. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from
Pseudomonas aeruginosa
. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the

K
m

was found to be 4.6 × 10
−4
M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.
Expression of the
fixR-nifA
Operon in
Bradyrhizobium japonicum
Depends on a New Response Regulator, RegR
Authors: Bauer Evelyne , Kaspar Thomas , Fischer Hans-Martin , Hennecke Hauke 
Published: December 2019

ABSTRACT

Many nitrogen fixation-associated genes in the soybean symbiont
Bradyrhizobium japonicum
are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the
fixR-nifA
operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension. This induction depends on a −24/−12-type promoter (
fixRp
1
) that is recognized by the ς
54
RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (
fixRp
2
) that overlaps with
fixRp
1
and depends on an upstream DNA region (UAS) located around position −68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760–1765, 1995). A protein binding to the UAS was previously postulated to act as an activator. This protein has now been purified, and the corresponding gene (
regR
) has been cloned. On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems. We identified upstream of the
regR
gene an additional gene (
regS
) encoding a putative sensor kinase. A
regR
mutant was constructed in which neither a specific UAS-binding activity nor
fixRp
2
-dependent transcript formation and
fixR′-′lacZ
expression was detected in aerobically grown cells. Anaerobic
fixR′-′lacZ
expression was also decreased in
regR
mutants to about 10% of the level observed in the wild type. Similarly,
regR
mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by
nifA
mutants, the morphology of those nodules was normal, displaying no signs of necrosis. While
regR
mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of
regS
mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains. The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling
nif
and
fix
gene expression in
B. japonicum
.
Activation Control of
pur
Gene Expression in
Lactococcus lactis
: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of
purC
and
purD
Promoter Regions
Authors: Kilstrup Mogens , Jessing Stine G. , Wichmand-Jørgensen Stephanie B. , Madsen Mette , Nilsson Dan 
Published: December 2019

ABSTRACT

A comparison of the
purC
and
purD
upstream regions from
Lactococcus lactis
revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς
70
promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in
purC
is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the
purC
and
purD
promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea that
purC
and
purD
transcription is regulated by a transcriptional activator binding to the PurBox sequence.
Structural and Biochemical Analysis of the Sheath of
Phormidium uncinatum
Authors: Hoiczyk Egbert 
Published: December 2019

ABSTRACT

The sheath of the filamentous, gliding cyanobacterium
Phormidium uncinatum
was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in
P. uncinatum
directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699–708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In
P. uncinatum
the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.
The Flagellar Switch Genes
fliM
and
fliN
of
Rhodobacter sphaeroides
Are Contained in a Large Flagellar Gene Cluster
Authors: García Norma , Campos Andrés , Osorio Aurora , Poggio Sebastian , González-Pedrajo Bertha , Camarena Laura , Dreyfus Georges 
Published: December 2019

ABSTRACT

In this work, the genes that encode the FliM and FliN proteins of
Rhodobacter sphaeroides
were characterized. These genes are part of a large flagellar gene cluster in which six additional open reading frames encoding products homologous to FliL, FliO, FliP, FliQ, FliR, and FlhB proteins from other bacteria were identified. The inactivation of the
fliM
gene gave a nonflagellate phenotype (Fla
−
), suggesting that FliM is required for flagellar assembly. Complementation analysis of this
fliM
mutant indicated that
fliM
and
fliN
transcription starts beyond the 5′ end of
fliK
and terminates after
fliN
.
Construction and Physiological Analysis of a
Xanthomonas
Mutant To Examine the Role of the
oxyR
Gene in Oxidant-Induced Protection against Peroxide Killing
Authors: Mongkolsuk Skorn , Sukchawalit Rojana , Loprasert Suvit , Praituan Wipa , Upaichit Apichat 
Published: December 2019

ABSTRACT

We constructed and characterized a
Xanthomonas campestris
pv. phaseoli
oxyR
mutant. The mutant was hypersensitive to H
2
O
2
and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and
ahpC
genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the
oxyR
mutant. These results show that
X. campestris
pv. phaseoli
oxyR
is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.
Cloning, Characterization, and Transcriptional Analysis of a Gene Encoding an α-Crystallin-Related, Small Heat Shock Protein from the Thermophilic Cyanobacterium
Synechococcus vulcanus
Authors: Roy Sanjit Kumer , Nakamoto Hitoshi 
Published: December 2019

ABSTRACT

hspA
, a gene encoding a 16-kDa heat-induced protein from the thermophilic cyanobacterium
Synechococcus vulcanus
, has been cloned and sequenced. The deduced amino acid sequence of the gene product showed significant homology to sequences of the family of α-crystallin-related, small heat shock proteins. A monocistronic mRNA of
hspA
increased transiently in response to heat shock. The heat shock induction occurred at a vegetative promoter but without the CIRCE (controlling inverted repeat of chaperone expression) element.
A Silent ABC Transporter Isolated from
Streptomyces rochei
F20 Induces Multidrug Resistance
Authors: Fernández-Moreno Miguel A. , Carbó Lázaro , Cuesta Trinidad , Vallín Carlos , Malpartida Francisco 
Published: December 2019

ABSTRACT

In the search for heterologous activators for actinorhodin production in
Streptomyces lividans
, 3.4 kb of DNA from
Streptomyces rochei
F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the
actII-ORF4
gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3′ end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3′ terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named
msr
, is a new bacterial multidrug ABC transporter.
Molecular Cloning and Functional Expression of a Protein-Serine/Threonine Phosphatase from the Hyperthermophilic Archaeon
Pyrodictium abyssi
TAG11
Authors: Mai Bianca , Frey Gerhard , Swanson Ronald V. , Mathur Eric J. , Stetter K. O. 
Published: December 2019

ABSTRACT

An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon
Pyrodictium abyssi
TAG11 and named Py-PP1. Py-PP1 was expressed in
Escherichia coli
, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo.