bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Two Genes from
Streptomyces argillaceus
Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin
Authors: Fernández Ernestina , Weißbach Ulrike , Reillo César Sánchez , Braña Alfredo F. , Méndez Carmen , Rohr Jürgen , Salas José A. 
Published: December 2019

ABSTRACT

Mithramycin is an antitumor polyketide drug produced by
Streptomyces argillaceus
that contains two deoxysugar chains, a disaccharide consisting of two
d
-olivoses and a trisaccharide consisting of a
d
-olivose, a
d
-oliose, and a
d
-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb
Pst
I-
Xho
I fragment was sequenced, and two divergent genes (
mtmGI
and
mtmGII
) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by
mtmGI
and
mtmGII
are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.
Influence of Lif, the Lysostaphin Immunity Factor, on Acceptors of Surface Proteins and Cell Wall Sorting Efficiency in
Staphylococcus carnosus
Authors: Strauss Andreas , Thumm Günther , Götz Friedrich 
Published: December 2019

ABSTRACT

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of
Staphylococcus simulans
was expressed in
Staphylococcus carnosus
, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.
Flk Couples
flgM
Translation to Flagellar Ring Assembly in
Salmonella typhimurium
Authors: Karlinsey Joyce E. , Tsui Ho-Ching T. , Winkler Malcolm E. , Hughes Kelly T. 
Published: December 2019

ABSTRACT

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in
Salmonella typhimurium
. The FlgM protein inhibits the flagellum-specific transcription factor ς
28
in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The
flk
gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of ς
28
-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (
flgA
,
flgH
, and
flgI
) by introduction of a null mutation in the
flk
gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389–2400, 1997). In P- and L-ring mutant strains, recessive mutations in
flk
resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla
+
) strains. The reduction in intracellular FlgM levels by mutations in the
flk
gene was concomitant with a 10-fold increase in transcription of the
flgMN
operon compared to that of the isogenic
flk
+
strain, while transcription of the
flgAMN
operon was unaffected. This was true for both direct measurement of the
flgAMN
and
flgMN
mRNA transcripts by RNase T2 protection assays and for
lac
operon fusions to either the
flgAMN
or
flgMN
promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occured primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With
lacZ
translational fusions to
flgM
, a null mutation in
flk
resulted in a significant reduction of
flgM-lacZ
mRNA translation, expressed from the class 3
flgMN
promoter, in P- and L-ring mutant strains. No reduction in either
flgAMN
or
flgMN
mRNA stability was measured in the absence of Flk in Fla
+
, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the
flk
gene is only at the level of
flgM
mRNA translation.
Molecular Cloning and Expression Analysis of the
Rhodobacter capsulatus sodB
Gene, Encoding an Iron Superoxide Dismutase
Authors: Cortez Néstor , Carrillo Néstor , Pasternak Cécile , Balzer Angelika , Klug Gabriele 
Published: December 2019

ABSTRACT

Genetic complementation of a
sodA sodB Escherichia coli
mutant strain was used to clone
Rhodobacter capsulatus
genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The
R. capsulatus sodB
gene was expressed in
E. coli
, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared
Rhodobacter
lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The
sodB
gene was expressed at low levels in
R. capsulatus
cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a
sodB
mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated
sodB
gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of
R. capsulatus
in aerobic cultures.
Influence of the MexAB-OprM Multidrug Efflux System on Quorum Sensing in
Pseudomonas aeruginosa
Authors: Evans Kelly , Passador Luciano , Srikumar Ramakrishnan , Tsang Eric , Nezezon Jonathon , Poole Keith 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa nalB
mutants which hyperexpress the MexAB-OprM multidrug efflux system produce reduced levels of several extracellular virulence factors known to be regulated by quorum sensing. Such mutants also produce less acylated homoserine lactone autoinducer PAI-1, consistent with an observed reduction in
lasI
expression. These data suggest that PAI-1 is a substrate for MexAB-OprM, and its resulting exclusion from cells hyperexpressing MexAB-OprM limits PAI-1-dependent activation of
lasI
and the virulence genes.
A Novel Mechanism for Resistance to the Antimetabolite N -Phosphonoacetyl-
l
-Aspartate by
Helicobacter pylori
Authors: Burns Brendan P. , Mendz George L. , Hazell Stuart L. 
Published: December 2019

ABSTRACT

The mechanism of resistance to
N
-phosphonoacetyl-
l
-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in
Helicobacter pylori
was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of
H. pylori
. The inhibitor was taken up by
H. pylori
cells and the transport was saturable, with a

K
m

of 14.8 mM and a
V
max
of 19.1 nmol min
−1
μl of cell water
−1
. By
31
P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of
H. pylori
studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by
1
H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when
N
-acetyl-
l
-aspartate was incubated with
H. pylori
lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two
H. pylori
enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.
Posttranscriptional Control Mediates Cell Type-Specific Localization of Catalase A during
Aspergillus nidulans
Development
Authors: Navarro Rosa E. , Aguirre Jesús 
Published: December 2019

ABSTRACT

Two differentially regulated catalase genes have been identified in the fungus
Aspergillus nidulans
. The
catA
gene belongs to a class whose transcripts are specifically induced during asexual sporulation (conidiation) and encodes a catalase accumulated in conidia. Using a developmental mutant affected in the
brlA
gene, which is unable to form conidia but capable of producing sexual spores (ascospores), we demonstrated that the
catA
mRNA accumulated during induction of conidiation but did not produce CatA protein. In contrast, high levels of catalase A activity were detected in the ascospores produced by this mutant, indicating that the
catA
gene is posttranscriptionally regulated. The same type of regulation was observed for a
catA
::
lacZ
translational gene fusion, suggesting that the
catA
message 5′ untranslated region could be involved in translational control during development. In a wild-type strain, β-galactosidase activity driven from the
catA
::
lacZ
gene fusion was low in hyphae and increased 50-fold during conidiation and 620-fold in isolated conidia. Consistent with this finding spatial expression of the reporter gene was restricted to metulae, phialides, and conidia. Conidium-associated expression was maintained in a
stuA
mutant, in which the conidiophore cell pattern is severely deranged.
catA
mRNA accumulation was also observed when vegetative mycelia was subject to oxidative, osmotic, and nitrogen or carbon starvation stress. Nevertheless, catalase A activity was restricted to the conidia produced under nutrient starvation. Our results provide support for a model in which translation of the
catA
message, accumulated during conidiation or in response to different types of stress, is linked to the morphogenetic processes involved in asexual and sexual spore formation. Our findings also indicate that
brlA
-independent mechanisms regulate the expression of genes encoding spore-specific products.
Haemophilus ducreyi
Secretes a Filamentous Hemagglutinin-Like Protein
Authors: Ward Christine K. , Lumbley Sheryl R. , Latimer Jo L. , Cope Leslie D. , Hansen Eric J. 
Published: December 2019

ABSTRACT

We have identified two extremely large open reading frames (ORFs) in
Haemophilus ducreyi
35000,
lspA1
and
lspA2
, each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of
Bordetella pertussis
filamentous hemagglutinin (FhaB). To the best of our knowledge,
lspA1
(12,500 nucleotides [nt]) and
lspA2
(14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that
lspA1
and
lspA2
sequences were present in 15 other geographically diverse
H. ducreyi
strains. Reverse transcriptase PCR analysis of total RNA isolated from
H. ducreyi
35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of
H. ducreyi
-infected rabbits indicated that
lspA1
and
lspA2
were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulent
H. ducreyi
strains reacted with both polyclonal serum from rabbits infected with
H. ducreyi
35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in
H. ducreyi
35000 culture supernatant was shown to be the protein product of the
lspA1
ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four
H. ducreyi
strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of
H. ducreyi
to cause lesions in this animal model.
A Promoter Relay Mechanism for Sequential Gene Activation
Authors: Fang Ming , Wu Hai-Young 
Published: December 2019

ABSTRACT

The effect of DNA supercoiling on gene expression is dependent not only on specific genes but also on the sequence context of the genes. This position-dependent supercoiling effect on gene activation is best illustrated in the study of the suppression of the
leu-500
mutation of the
leuABCD
operon in a
Salmonella typhimurium topA
mutant. In this communication, we report a novel promoter relay mechanism whereby several genes are sequentially expressed in a position-dependent manner: the
ilvIH
promoter (
pilvIH
) activates a cryptic
leuO
promoter (
pleuO
) located between the two divergently arrayed
ilvIH
and
leu-500
promoters. Both the
cis
-acting
pleuO
activity and the
trans
-acting LeuO protein are necessary for subsequent activation of the
leu-500
promoter (
pleu-500
). Furthermore,
pleuO
can be functionally replaced with the inducible
tac
promoter (
ptac
) for
leu-500
activation, suggesting that transcription-driven DNA supercoiling underlies the relay mechanism. This is the first example of several related genes communicating via a promoter relay mechanism for their coordinated expression.
Constricted Flux through the Branched-Chain Amino Acid Biosynthetic Enzyme Acetolactate Synthase Triggers Elevated Expression of Genes Regulated by
rpoS
and Internal Acidification
Authors: Van Dyk Tina K. , Ayers Brenda L. , Morgan Robin W. , Larossa Robert A. 
Published: December 2019

ABSTRACT

The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism,
Escherichia coli
genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of random
E. coli
DNA fragments to
Photorhabdus luminescens luxCDABE
were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-
lux
fusions were identified. The
lux
reporter genes were joined to seven uncharacterized open reading frames,
f253a
,
f415
,
frvX
,
o513
,
o521
,
yciG
, and
yohF
, and five known genes,
inaA
,
ldcC
,
osmY
,
poxB
, and
sohA
. Inactivation of the
rpoS
-encoded sigma factor, ς
S
, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the ς
S
regulon,
f253a
,
ldcC
,
yciG
, and
yohF
, and included two known members,
osmY
and
poxB
. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-
lux
fusion-containing strains, namely, those with fusions of the ς
S
-controlled genes and
inaA
. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the ς
S
-dependent stress response.
Molecular Cloning and Characterization of Fengycin Synthetase Gene
fenB
from
Bacillus subtilis
Authors: Lin Guang-Huey , Chen Chyi-Liang , Tschen Johannes Scheng-Ming , Tsay San-San , Chang Yu-Sun , Liu Shih-Tung 
Published: December 2019

ABSTRACT

A fengycin synthetase gene,
fenB
, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in
Escherichia coli
and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25°C, an optimum pH of 4.5, a

K
m

value of 922 μM, and a turnover number of 236 s
−1
. FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.
Iron Uptake inUstilago maydis: Tracking the Iron Path
Authors: Ardon Orly , Nudelman Raphael , Caris Catherine , Libman Jacqueline , Shanzer Abraham , Chen Yona , Hadar Yitzhak 
Published: December 2019

ABSTRACTIn this study, we monitored and compared the uptake of iron in the fungusUstilago maydisby using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.
FtsZ Dynamics during the Division Cycle of Live
Escherichia coli
Cells
Authors: Sun Qin , Margolin William 
Published: December 2019

ABSTRACT

The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing
Escherichia coli
cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.
Mosaic Structure of the
smpB-nrdE
Intergenic Region of
Salmonella enterica
Authors: Bäumler Andreas J. , Heffron Fred 
Published: December 2019

ABSTRACT

The
Salmonella enterica smpB-nrdE
intergenic region contains about 45 kb of DNA that is not present in
Escherichia coli
. This DNA region was not introduced by a single horizontal transfer event, but was generated by multiple insertions and/or deletions that gave rise to a mosaic structure in this area of the chromosome.
The 102-Kilobase Unstable Region of
Yersinia pestis
Comprises a High-Pathogenicity Island Linked to a Pigmentation Segment Which Undergoes Internal Rearrangement
Authors: Buchrieser Carmen , Prentice Michael , Carniel Elisabeth 
Published: December 2019

ABSTRACT

Several pathogenicity islands have recently been identified in different bacterial species, including a high-pathogenicity island (HPI) in
Yersinia enterocolitica
1B. In
Y. pestis
, a 102-kb chromosomal fragment (
pgm
locus) that carries genes involved in iron acquisition and colony pigmentation can be deleted en bloc. In this study, characterization and mapping of the 102-kb region of
Y. pestis
6/69 were performed to determine if this unstable region is a pathogenicity island. We found that the 102-kb region of
Y. pestis
is composed of two clearly distinct regions: an ≈35-kb iron acquisition segment, which is an HPI per se, linked to an ≈68-kb pigmentation segment. This linkage was preserved in all of the
Y. pestis
strains studied. However, several nonpigmented
Y. pestis
strains harboring an
irp2
gene have been previously identified, suggesting that the pigmentation segment is independently mobile. Comparison of the physical map of the 102-kb region of these strains with that of strain 6/69 and complementation experiments were carried out to determine the genetic basis of this phenomenon. We demonstrate that several different mechanisms involving mutations and various-size deletions are responsible for the nonpigmented phenotype in the nine strains studied. However, no deletion corresponded exactly to the pigmentation segment. The 102-kb region of
Y. pestis
is an evolutionarily stable linkage of an HPI with a pigmentation segment in a region of the chromosome prone to rearrangement in vitro.