bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Potential Cell Wall Glucanases of
Saccharomyces cerevisiae
and Their Involvement in Mating
Authors: Cappellaro Corinna , Mrsa Vladimir , Tanner Widmar 
Published: December 2019

ABSTRACT

Biotinylation of intact
Saccharomyces cerevisiae
cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT). By N-terminal sequencing, three of these proteins were identified as the known proteins β-exoglucanase 1 (Exg1p), β-endoglucanase (Bgl2p), and chitinase (Cts1p). One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p [for soluble cell wall proteins]) were found to be related to glucanases. Single knockouts of these three potential glucanases did not result in dramatic phenotypes. The double knockout of
SCW4
and the homologous gene
SCW10
resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types. The synergistic behavior of the disruption of
SCW4
and
SCW10
was partly antagonized by the disruption of
BGL2
. The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities.
Induction of Ca
2+
-Calmodulin Signaling by Hard-Surface Contact Primes
Colletotrichum gloeosporioides
Conidia To Germinate and Form Appressoria
Authors: Kim Yeon-Ki , Li Daoxin , Kolattukudy Pappachan E. 
Published: December 2019

ABSTRACT

Hard-surface contact primes the conidia of
Colletotrichum gloeosporioides
to respond to plant surface waxes and a fruit-ripening hormone, ethylene, to germinate and form the appressoria required for infection of the host. Our efforts to elucidate the molecular events in the early phase of the hard-surface contact found that EGTA (5 mM) and U73122 (16 nM), an inhibitor of phospholipase C, inhibited (50%) germination and appressorium formation. Measurements of calmodulin (CaM) transcripts with a CaM cDNA we cloned from
C. gloeosporioides
showed that CaM was induced by hard-surface contact maximally at 2 h and then declined; ethephon enhanced this induction. The CaM antagonist, compound 48/80, completely inhibited conidial germination and appressorium formation at a concentration of 3 μM, implying that CaM is involved in this process. A putative CaM kinase (CaMK) cDNA of
C. gloeosporioides
was cloned with transcripts from hard-surface-treated conidia. A selective inhibitor of CaMK, KN93 (20 μM), inhibited (50%) germination and appressorium formation, blocked melanization, and caused the formation of abnormal appressoria. Scytalone, an intermediate in melanin synthesis, reversed the inhibition of melanization but did not restore appressorium formation. The phosphorylation of 18- and 43-kDa proteins induced by hard-surface contact and ethephon was inhibited by the treatment with KN93. These results strongly suggest that hard-surface contact induces Ca
2+
-calmodulin signaling that primes the conidia to respond to host signals by germination and differentiation into appressoria.
Degradation of 2,4,6-Trichlorophenol by
Phanerochaete chrysosporium
: Involvement of Reductive Dechlorination
Authors: Reddy G. Vijay Bhasker , Sollewijn Gelpke Maarten D. , Gold Michael H. 
Published: December 2019

ABSTRACT

Under secondary metabolic conditions, the lignin-degrading basidiomycete
Phanerochaete chrysosporium
mineralizes 2,4,6-trichlorophenol. The pathway for the degradation of 2,4,6-trichlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway is initiated by a LiP- or MnP-catalyzed oxidative dechlorination reaction to produce 2,6-dichloro-1,4-benzoquinone. The quinone is reduced to 2,6-dichloro-1,4-dihydroxybenzene, which is reductively dechlorinated to yield 2-chloro-1,4-dihydroxybenzene. The latter is degraded further by one of two parallel pathways: it either undergoes further reductive dechlorination to yield 1,4-hydroquinone, which is
ortho
-hydroxylated to produce 1,2,4-trihydroxybenzene, or is hydroxylated to yield 5-chloro-1,2,4-trihydroxybenzene, which is reductively dechlorinated to produce the common key metabolite 1,2,4-trihydroxybenzene. Presumably, the latter is ring cleaved with subsequent degradation to CO
2
. In this pathway, the chlorine at C-4 is oxidatively dechlorinated, whereas the other chlorines are removed by a reductive process in which chlorine is replaced by hydrogen. Apparently, all three chlorine atoms are removed prior to ring cleavage. To our knowledge, this is the first reported example of aromatic reductive dechlorination by a eukaryote.
Roles of the
Escherichia coli
Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo
Authors: Thomas Jeffrey G. , Baneyx François 
Published: December 2019

ABSTRACT

We have constructed an
Escherichia coli
strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the
clpA
,
clpB
, or
htpG
gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the
clpA
,
htpG
, and
ibp
null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δ
ibp
led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes
clpB
and
htpG
resulted in growth defects at 42°C when combined with the
dnaK756
or
groES30
alleles, while the Δ
ibp
mutation had a detrimental effect only on the growth of
dnaK756
mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of
dnaK756
or
groES30
cells at high temperatures. Whereas increased levels of host protein aggregation were observed in
dnaK756
and
groES30
mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved
E. coli
small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major
E. coli
chaperone systems in vivo.
HrpW of
Erwinia amylovora
, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class
Authors: Kim Jihyun F. , Beer Steven V. 
Published: December 2019

ABSTRACT

Harpins, such as HrpN of
Erwinia amylovora
, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified
hrpW
of
E. amylovora
, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of
hrpW
, and Western blot analysis of
hrpL
mutants indicated that the production of HrpW is regulated by
hrpL
. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and
hrp
secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of
E. amylovora
consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that
hrpW
is conserved among several
Erwinia
species, and
hrpW
, provided in
trans
, enhanced the HR-inducing ability of a
hrpN
mutant. However, HrpW did not increase the virulence of a
hrpN
mutant in host tissue, and
hrpW
mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.
Carbon-Source-Dependent Expression of the
PalkB
Promoter from the
Pseudomonas oleovorans
Alkane Degradation Pathway
Authors: Yuste Luis , Canosa Inés , Rojo Fernando 
Published: December 2019

ABSTRACT

Pseudomonas oleovorans
GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein. In the presence of alkanes, AlkS activates the expression of promoter
PalkB
, from which most of the genes of the pathway are transcribed. In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the
alk
genes with the metabolic status of the cell. Reporter strains bearing
PalkB-lacZ
transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the
PalkB
promoter by a nonmetabolizable inducer. Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate. When cells were grown in Luria-Bertani rich medium,
PalkB
was strongly down-regulated. This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to
Escherichia coli
, a heterologous genetic background. Possible mechanisms responsible for
PalkB
regulation are discussed.
Identification of Multiple ς
54
-Dependent Transcriptional Activators in
Vibrio cholerae
Authors: Klose Karl E. , Novik Veronica , Mekalanos John J. 
Published: December 2019

ABSTRACT

In the pathogenic bacterium
Vibrio cholerae
, the alternate sigma factor ς
54
is required for expression of multiple sets of genes, including an unidentified gene(s) necessary for enhanced colonization within the host. To identify ς
54
-dependent transcriptional activators involved in colonization, PCR was performed with
V. cholerae
chromosomal DNA and degenerate primers, revealing six novel and distinct coding sequences with homology to ς
54
-dependent activators. One sequence had high homology to the
luxO
gene of
V. harveyi
, which in that organism is involved in quorum sensing. Phenotypes of
V. cholerae
strains containing mutations in each of the six putative ς
54
-dependent activator genes identified one as a probable
ntrC
homologue. None of the mutant strains exhibited a defect in the ability to colonize infant mice, suggesting the presence of additional ς
54
-dependent activators not identified by this technique.
Molecular Characterization of the Complete 23F Capsular Polysaccharide Locus of
Streptococcus pneumoniae
Authors: Ramirez Mario , Tomasz Alexander 
Published: December 2019

ABSTRACT

The complete DNA sequence of the capsular locus 23F of
Streptococcus pneumoniae
is presented. The 18.6-kb
cps23f
locus is composed of 18 open reading frames flanked at the 5′ and 3′ ends by the genes
dexB
and
aliA
, an arrangement similar to those of some of the other identified
cps
loci.
Detoxification of Protoanemonin by Dienelactone Hydrolase
Authors: Brückmann Markus , Blasco Rafael , Timmis Kenneth Nigel , Pieper Dietmar Helmut 
Published: December 2019

ABSTRACT

Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of
Pseudomonas
sp. strain B13 to
cis
-acetylacrylate. Although similar

K
m

values were observed for
cis
-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of
cis
-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The
trans
-dienelactone hydrolase of
Pseudomonas
sp. strain RW10 did not detectably transform protoanemonin. Obviously,
Pseudomonas
sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-
cis
,
cis
-muconate to the
cis
-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.
A Type IC Restriction-Modification System in
Lactococcus lactis
Authors: Schouler Catherine , Clier Florence , Lerayer Alda Luisa , Ehrlich S. Dusko , Chopin Marie-Christine 
Published: December 2019

ABSTRACT

Three genes coding for the endonuclease, methylase, and specificity subunits of a type I restriction-modification (R-M) system in the
Lactococcus lactis
plasmid pIL2614 have been characterized. Plasmid location, sequence homologies, and inactivation studies indicated that this R-M system is most probably of type IC.
Myxococcus xanthus
Displays Frz-Dependent Chemokinetic Behavior during Vegetative Swarming
Authors: Ward Mandy J. , Mok Kenny C. , Zusman David R. 
Published: December 2019

ABSTRACT

Myxococcus xanthus
has been shown to utilize both directed (tactic) and undirected (kinetic) movements during different stages of its complex life cycle. We have used time-lapse video microscopic analysis to separate tactic and kinetic behaviors associated specifically with vegetatively swarming cells. Isolated individual cells separated by a thin agar barrier from mature swarms showed significant increases in gliding velocity compared to that of similar cells some distance from the swarm. This orthokinetic behavior was independent of the frequency of reversals of gliding direction (klinokinesis) but did require both the Frz signal transduction system and S-motility. We propose that
M. xanthus
uses Frz-dependent, auto-orthokinetic behavior to facilitate the dispersal of cells under conditions where both cell density and nutrient levels are high.
Iron-Responsive Gene Regulation in a
Campylobacter jejuni fur
Mutant
Authors: van Vliet Arnoud H. M. , Wooldridge Karl G. , Ketley Julian M. 
Published: December 2019

ABSTRACT

The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe
2+
as a cofactor. Mutational analysis of the
Campylobacter jejuni fur
gene was carried out by generation of a set of mutant copies of
fur
which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the
C. jejuni
NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization.
C. jejuni
mutants were obtained only when the resistance genes were transcribed in the same orientation as the
fur
gene. The
C. jejuni fur
mutant grew slower than the parental strain. Comparison of protein profiles of fractionated
C. jejuni
cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a
Campylobacter coli
siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the
fur
mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the
C. jejuni
iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.
Identification of
Candida albicans ALS2
and
ALS4
and Localization of Als Proteins to the Fungal Cell Surface
Authors: Hoyer L. L. , Payne T. L. , Hecht J. E. 
Published: December 2019

ABSTRACT

Additional genes in the growing
ALS
family of
Candida albicans
were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of
ALS1
. This procedure yielded fosmids encoding
ALS2
and
ALS4. ALS2
and
ALS4
conformed to the three-domain structure of
ALS
genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in
Saccharomyces cerevisiae
demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other
ALS
genes,
ALS2
and
ALS4
were differentially regulated.
ALS4
expression was correlated with the growth phase of the culture;
ALS2
expression was not observed under many different in vitro growth conditions. The data presented here demonstrate that
ALS
genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on the
C. albicans
cell surface vary with strain and growth conditions.
Novel
rkp
Gene Clusters of
Sinorhizobium meliloti
Involved in Capsular Polysaccharide Production and Invasion of the Symbiotic Nodule: the
rkpK
Gene Encodes a UDP-Glucose Dehydrogenase
Authors: Kereszt Attila , Kiss Ernő , Reuhs Bradley L. , Carlson Russell W. , Kondorosi Ádám , Putnoky Péter 
Published: December 2019

ABSTRACT

The production of exopolysaccharide (EPS) was shown to be required for the infection process by rhizobia that induce the formation of indeterminate nodules on the roots of leguminous host plants. In
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
) Rm41, a capsular polysaccharide (KPS) analogous to the group II K antigens of
Escherichia coli
can replace EPS during symbiotic nodule development and serve as an attachment site for the strain-specific bacteriophage φ16-3. The
rkpA
to -
J
genes in the chromosomal
rkp-1
region code for proteins that are involved in the synthesis, modification, and transfer of an as-yet-unknown lipophilic molecule which might function as a specific lipid carrier during KPS biosynthesis. Here we report that with a phage φ16-3-resistant population obtained after random Tn
5
mutagenesis, we have identified novel mutants impaired in KPS production by genetic complementation and biochemical studies. The mutations represent two novel loci, designated the
rkp-2
and
rkp-3
regions, which are required for the synthesis of rhizobial KPS. The
rkp-2
region harbors two open reading frames (ORFs) organized in monocistronic transcription units. Although both genes are required for normal lipopolysaccharide production, only the second one, designated
rkpK
, is involved in the synthesis of KPS. We have demonstrated that RkpK possesses UDP-glucose dehydrogenase activity, while the protein product of ORF1 might function as a UDP-glucuronic acid epimerase.
The 20S Proteasome of
Streptomyces coelicolor
Authors: Nagy István , Tamura Tomohiro , Vanderleyden Jos , Baumeister Wolfgang , De Mot René 
Published: December 2019

ABSTRACT

20S proteasomes were purified from
Streptomyces coelicolor
A3(2) and shown to be built from one α-type subunit (PrcA) and one β-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the
Streptomyces
metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (
prcBA
) similar to
Rhodococcus
and
Mycobacterium
spp. and showed that the β subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved
orf7
and an AAA ATPase gene (
arc
).
Efficiency of the pTF-FC2
pas
Poison-Antidote Stability System in
Escherichia coli
Is Affected by the Host Strain, and Antidote Degradation Requires the Lon Protease
Authors: Smith Anthony S. G. , Rawlings Douglas E. 
Published: December 2019

ABSTRACT

The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (
pas
) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in
Escherichia coli
CSH50, a 2.5-fold increase in
E. coli
JM105, and no detectable stabilization in
E. coli
strains JM107 and JM109. The lethality of the PasB toxin was far higher in the
E. coli
strains in which the
pas
was most effective. Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin. A decrease in host cell death following plasmid loss from an
E. coli lon
mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.
Functional Consequences of Changing Proline Residues in the Phenylalanine-Specific Permease of
Escherichia coli
Authors: Pi Jing , Dogovski C. , Pittard A. J. 
Published: December 2019

ABSTRACT

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium
Escherichia coli
. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650–2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [
35
S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.