bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Genes Encoding
d
-Amino Acid Transaminase and Glutamate Racemase, Two
d
-Glutamate Biosynthetic Enzymes of
Bacillus sphaericus
ATCC 10208
Authors: Fotheringham Ian G. , Bledig Stefan A. , Taylor Paul P.
Published: December 2019

In
Bacillus sphaericus
and other
Bacillus
spp.,
d
-amino acid transaminase has been considered solely responsible for biosynthesis of
d
-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the
dat
gene encoding
d
-amino acid transaminase and the
glr
gene encoding a glutamate racemase from
B. sphaericus
ATCC 10208. The
glr
gene encodes a 28.8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of
Lactobacillus
,
Pediococcus
, and
Staphylococcus
species. The
dat
gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the
d
-amino acid transaminase of the thermophilic
Bacillus
sp. strain YM1.
Complete Nucleotide Sequence of pSK41: Evolution of Staphylococcal Conjugative Multiresistance Plasmids
Authors: Berg Tracey , Firth Neville , Apisiridej Sumalee , Hettiaratchi Anusha , Leelaporn Amornrut , Skurray Ronald A.
Published: December 2019

The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from
Staphylococcus aureus
, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn
552
-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS
257
-mediated cointegrative capture of other plasmids.
A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in
Haemophilus influenzae
Rd
Authors: Macfadyen Leah P. , Ma Caixia , Redfield Rosemary J.
Published: December 2019

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in
Haemophilus influenzae
. In
Escherichia coli
, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the
cpdA
gene. We have used several approaches to demonstrate that the homologous
icc
gene of
H. influenzae
encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In
E. coli
, expression of cloned
icc
reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for
cpdA
. In
H. influenzae
, an
icc
null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a
cya
mutation, an
icc
strain was 10,000-fold more sensitive to exogenous cAMP than an
icc
+
strain. The
icc
strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a
cya
strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the
H. influenzae
cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.
Contribution of the P

mra

Promoter to Expression of Genes in the
Escherichia coli mra
Cluster of Cell Envelope Biosynthesis and Cell Division Genes
Authors: Mengin-Lecreulx Dominique , Ayala Juan , Bouhss Ahmed , van Heijenoort Jean , Parquet Claudine , Hara Hiroshi
Published: December 2019

Recently, a promoter for the essential gene
ftsI
, which encodes penicillin-binding protein 3 of
Escherichia coli
, was precisely localized 1.9 kb upstream from this gene, at the beginning of the
mra
cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (P

mra

) on the chromosome and its replacement by the
lac
promoter (P

mra

::P

lac

) led to isopropyl-β-
d
-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P

mra

to
ftsW
, the fifth gene downstream from
ftsI
, was provided in
trans
on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P

mra

::P

lac

promoter expression was repressed or fully induced. It was confirmed that the P

mra

promoter is required for expression of the first nine genes of the
mra
cluster:
mraZ
(
orfC
),
mraW
(
orfB
),
ftsL
(
mraR
),
ftsI
,
murE
,
murF
,
mraY
,
murD
, and
ftsW
. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P

mra

::P

lac

cells. This was correlated with an accumulation of the nucleotide precursors UDP–
N
-acetylglucosamine and UDP–
N
-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–
N
-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of
ftsZ
, the penultimate gene from this cluster, was significantly reduced when P

mra

expression was repressed. It was concluded that the transcription of the genes located downstream from
ftsW
in the
mra
cluster, from
murG
to
ftsZ
, is also mainly (but not exclusively) dependent on the P

mra

promoter.
Unique Regulation of Carbohydrate Chemotaxis in
Bacillus subtilis
by the Phosphoenolpyruvate-Dependent Phosphotransferase System and the Methyl-Accepting Chemotaxis Protein McpC
Authors: Garrity Liam F. , Schiel Stacey L. , Merrill Ronald , Reizer Jonathan , Saier Milton H. , Ordal George W.
Published: December 2019

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of
Escherichia coli
to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in
Bacillus subtilis
is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for
E. coli
, a
B. subtilis ptsH
null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An
mcpC
null mutant showed no response to the stepwise addition or removal of
d
-glucose or
d
-mannitol, both of which are PTS substrates. Therefore, in contrast to
E. coli
PTS carbohydrate chemotaxis,
B. subtilis
PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate
d
-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that
B. subtilis
EnzymeI-P has the ability to inhibit
B. subtilis
CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of
d
-glucose, a decrease in
d
-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.
Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from
Streptomyces glaucescens
and Its Role in Initiation of Fatty Acid Biosynthesis
Authors: Han Lei , Lobo Sandra , Reynolds Kevin A.
Published: December 2019

The
Streptomyces glaucescens fabH
gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in
Escherichia coli
, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an
M
r
of 37,000, while gel filtration analysis determined a native
M
r
of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a

K
m

value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with

K
m

values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the
S. glaucescens
FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in
Streptomyces
and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in
S. glaucescens
.
Negative Regulation of
hrp
Genes in
Pseudomonas syringae
by HrpV
Authors: Preston Gail , Deng Wen-Ling , Huang Hsiou-Chen , Collmer Alan
Published: December 2019

Mutations in the five
hrp
and
hrc
genes in the
hrpC
operon of the phytopathogen
Pseudomonas syringae
pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The
hrcC
gene within the
hrpC
operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order),
hrpF
,
hrpG
, (
hrcC
),
hrpT
, and
hrpV
, appear to be unique to the group I
hrp
clusters found in certain phytopathogens (e.g.,
P. syringae
and
Erwinia amylovora
) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar
nptII
cartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions.
P. syringae
pv. syringae 61
hrpF
,
hrpG
, and
hrpT
mutants were unable to secrete HrpZ, whereas the
hrpV
mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the
hrpV
mutant produced higher levels of proteins encoded by all three of the major
hrp
operons tested—HrcJ (
hrpZ
operon), HrcC (
hrpC
operon), and HrcQ
B
(
hrpU
operon)—and that constitutive expression of
hrpV
in
trans
abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the
P. syringae
pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with ς
54
-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed
hrpV
to repress the activation of HrcJ production that normally accompanies constitutive expression of
hrpL
or
hrpRS
. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of the
hrpZ
operon was determined by monitoring the levels of β-glucuronidase produced by a
hrpA
′::
uidA
transcriptional fusion plasmid in different
P. syringae
pv. syringae 61 strains. The
hrpV
mutant produced higher levels of β-glucuronidase than the wild type, a
hrcU
(type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing
hrpV
in
trans
produced low levels equivalent to that of a
hrpS
mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.
Bacillus subtilis
Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth
Authors: Murray Thomas , Popham David L. , Setlow Peter
Published: December 2019

Bacillus subtilis
strains lacking penicillin-binding protein 1 (PBP1), encoded by
ponA
, required greater amounts of Mg
2+
or Ca
2+
for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of
ponA
cells in a medium low in Mg
2+
also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg
2+
to growth media eliminated these phenotypes of a
ponA
mutant. In contrast to the effects of divalent cations, NaCl did not restore
ponA
cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg
2+
addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and
ponA
cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.
Cloning and Identification of Conjugative Transfer Origins in the
Rhizobium meliloti
Genome
Authors: Herrera-Cervera Jose A. , Sanjuan-Pinilla Julio M. , Olivares Jose , Sanjuan Juan
Published: December 2019

A simple approach was used to identify
Rhizobium meliloti
DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (
oriT
,
mob
). RecA-defective
R. meliloti
merodiploid populations, where each individual contained a hybrid cosmid from an
R. meliloti
GR4 gene library, were used as donors en masse in conjugation with another
R. meliloti
recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential
oriT
s. Individual hybrid cosmids were confirmed to be mobilized from the original
recA
donors at frequencies ranging from 10
−2
to 10
−5
per recipient cell. DNA hybridization experiments showed that seven
mob
DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by
R. meliloti
GR4. Another three
mob
clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the
oriT
of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for
oriT
s. Moreover, transfer of the 11
mob
cosmids from
R. meliloti
into
Escherichia coli
occurred at frequencies as high as 10
−1
, demonstrating the
R. meliloti
gene transfer capacity is not limited to the family
Rhizobiaceae
. Our results show that the
R. meliloti
genome contains multiple
oriT
s that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.
SdeK Is Required for Early Fruiting Body Development in
Myxococcus xanthus
Authors: Garza Anthony G. , Pollack Jeffrey S. , Harris Baruch Z. , Lee Albert , Keseler Ingrid M. , Licking Ellen F. , Singer Mitchell
Published: December 2019

Myxococcus xanthus
cells carrying the Ω4408 Tn
5lac
insertion at the
sde
locus show defects in fruiting body development and sporulation. Our analysis of
sde
expression patterns showed that this locus is induced early in the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h of development. Further studies showed that expression of
sde
is induced as growing cells enter stationary phase, suggesting that activation of the
sde
locus is not limited to the developmental process. Because the peak levels of
sde
expression in both an
sde
+
and an
sde
mutant background were similar, we conclude that the
sde
locus is not autoregulated. Characterization of the
sde
locus by DNA sequence analysis indicated that the Ω4408 insertion occurred within the
sdeK
gene. Primer extension analyses localized the 5′ end of
sde
transcript to a guanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequence in the −12 and −24 regions upstream of the
sde
transcriptional start site shows similarity to the ς
54
family of promoters. The results of complementation studies suggest that the defects in development and sporulation caused by the Ω4408 insertion are due to an inactivation of
sdeK
. The predicted amino acid sequence of SdeK was found to have similarity to the sequences of the histidine protein kinases of two-component regulatory systems. Based on our results, we propose that SdeK may be part of a signal transduction pathway required for the activation and propagation of the early developmental program.
Functional Dissection of the Molybdate-Responsive Transcription Regulator, ModE, from
Escherichia coli
Authors: McNicholas Paul M. , Mazzotta Mandy M. , Rech Sabine A. , Gunsalus Robert P.
Published: December 2019

The product of the
Escherichia coli modE
gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the
modA
promoter in vitro and to repress expression from a
modA-lacZ
operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of λ CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the
moaA
and
dmsA
promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.
Analysis of the Exochelin Locus in
Mycobacterium smegmatis
: Biosynthesis Genes Have Homology with Genes of the Peptide Synthetase Family
Authors: Yu Shengwei , Fiss Ellen , Jacobs William R.
Published: December 2019

Mycobacteria secrete the siderophore exochelin when grown under iron-limiting conditions. In order to understand iron uptake mechanisms in mycobacteria, we have taken a genetic approach to identify those genes involved in exochelin biosynthesis and transport in
Mycobacterium smegmatis
. Of the 6,000 chemically mutagenized clones of
M. smegmatis
mc
2
155 screened on agar plates containing chrome azural S, 19 mutants that had lost the ability to produce or secrete exochelin were identified. Thirteen of these mutants were complemented by a single
M. smegmatis
cosmid. Sequence analysis of this cosmid revealed nine open reading frames, three of which are homologous to genes encoding transporter proteins, which are likely involved in exochelin transport. Complementation and Tn
10
mutagenesis analysis identified two new genes,
fxbB
and
fxbC
, which are required for exochelin biosynthesis. The
fxbB
and
fxbC
genes encode large proteins of 257 and 497 kDa, respectively, which are highly homologous to peptide synthetases, indicating that exochelin biosynthesis occurs by a nonribosomal mechanism.
Mutations in
toxR
and
toxS
That Separate Transcriptional Activation from DNA Binding at the Cholera Toxin Gene Promoter
Authors: Pfau James D. , Taylor Ronald K.
Published: December 2019

ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in
Vibrio cholerae
. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12
toxR
and
toxS
positive control mutants producing variant ToxR proteins from the
toxRS
operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in
toxR
specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven
toxR
mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion of
toxS
did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.
Mutational Inactivation of a Gene Homologous to
Escherichia coli ptsP
Affects Poly-β-Hydroxybutyrate Accumulation and Nitrogen Fixation in
Azotobacter vinelandii
Authors: Segura Daniel , Espín Guadalupe
Published: December 2019

Strain DS988, an
Azotobacter vinelandii
mutant with a reduced capacity to accumulate poly-β-hydroxybutyrate, was isolated after mini-Tn
5
mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to
Escherichia coli ptsP
which encodes enzyme I
Ntr
, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the
ptsP
mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene
nifH
was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation in
ptsP
leads to a failure in poly-β-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.
A Natural Large Chromosomal Inversion in
Lactococcus lactis
Is Mediated by Homologous Recombination between Two Insertion Sequences
Authors: Daveran-Mingot Marie-Line , Campo Nathalie , Ritzenthaler Paul , Le Bourgeois Pascal
Published: December 2019

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related
Lactococcus lactis
subsp.
cremoris
strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS
905
element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS
905
element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the
oriC-terC
symmetry of the chromosome and the local genetic environment.
The
ggpS
Gene from
Synechocystis
sp. Strain PCC 6803 Encoding Glucosyl-Glycerol-Phosphate Synthase Is Involved in Osmolyte Synthesis
Authors: Marin Kay , Zuther Ellen , Kerstan Thomas , Kunert Anja , Hagemann Martin
Published: December 2019

A salt-sensitive mutant of
Synechocystis
sp. strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis. Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11. This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (
otsA
homolog)- and glycerol-3-phosphate-synthesizing enzymes. After construction and characterization of mutants defective in these genes, it became obvious that an
otsA
homolog (
sll
1566) (T. Kaneko et al., DNA Res. 3:109–136, 1996) encodes GGPS, since only the mutant affected in
sll
1566 showed salt sensitivity combined with a complete absence of GG accumulation. Furthermore, the overexpression of
sll
1566 in
Escherichia coli
led to the appearance of GGPS activity in the heterologous host. The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of
Synechocystis
.
Oxidative Stress Response and Characterization of theoxyR-ahpCandfurA-katGLoci inMycobacterium marinum
Authors: Pagán-Ramos E. , Song J. , McFalone M. , Mudd M. H. , Deretic V.
Published: December 2019

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized theoxyR-ahpCandfurA-katGloci in the nontuberculous pathogenMycobacterium marinum. In contrast toMycobacterium smegmatisand likeMycobacterium tuberculosisandMycobacterium leprae,M. marinumwas shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress responseoxyRand its subordinate geneahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to theoxyR-ahpCpromoter region fromM. marinumand additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence.M. marinumAhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region ofahpC. In contrast, OxyR did not bind to the sequences upstream of thekatGstructural gene, andkatGexpression did not follow the pattern seen withahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream ofkatGinM. marinum. ThefurA-katGlinkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences inahpCandkatGexpression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest thatM. marinumwill be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.
Spore Photoproduct Lyase from
Bacillus subtilis
Spores Is a Novel Iron-Sulfur DNA Repair Enzyme Which Shares Features with Proteins such as Class III Anaerobic Ribonucleotide Reductases and Pyruvate-Formate Lyases
Authors: Rebeil Roberto , Sun Yubo , Chooback Lilian , Pedraza-Reyes Mario , Kinsland Cynthia , Begley Tadhg P. , Nicholson Wayne L.
Published: December 2019

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of
Bacillus subtilis
spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned
B. subtilis splB
gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the
amyE
locus restored UV resistance to spores of a UV-sensitive mutant
B. subtilis
strain carrying a deletion-insertion mutation which removed the entire
splAB
operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant
B. subtilis
spores and from an
Escherichia coli
overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of
S
-adenosylmethionine as a cofactor.
Two Functionally Distinct Regions Upstream of the

cbb
I

Operon of
Rhodobacter sphaeroides
Regulate Gene Expression
Authors: Dubbs James M. , Tabita F. Robert
Published: December 2019

A number of

cbbF
I

::
lacZ
translational fusion plasmids containing various lengths of sequence 5′ to the form I (

cbb
I

) Calvin-Benson-Bassham cycle operon (

cbbF
I
cbbP
I
cbbA
I
cbbL
I
cbbS
I

) of
Rhodobacter sphaeroides
were constructed. Expression of β-galactosidase was monitored under a variety of growth conditions. It was found that 103 bp of sequence upstream of the

cbbF
I

transcription start was sufficient to confer low levels of regulated

cbb
I

promoter expression; this activity was dependent on the presence of an intact
cbbR
gene. Additionally,
R. sphaeroides
CbbR was shown to bind to the region between 9 and 100 bp 5′ to the

cbbF
I

transcription start. Inclusion of an additional upstream sequence, from 280 to 636 bp 5′ to

cbbF
I

, resulted in a significant increase in regulated

cbb
I

promoter expression under all growth conditions tested. A 50-bp region responsible for the majority of this increase occurs between 280 and 330 bp 5′ to

cbbF
I

. The additional 306 bp of upstream sequence from 330 to 636 bp also appears to play a positive regulatory role. A 4-bp deletion 281 to 284 bp 5′ to

cbbF
I

significantly reduced

cbb
I

expression while the proper regulatory pattern was retained. These studies provide evidence for the presence of two functionally distinct regions of the

cbb
I

promoter, with the distal domain providing significant regulated promoter activity that adheres to the normal pattern of expression.
Transcriptional Analysis and Mutation of a
dnaA
-Like Gene in
Synechocystis
sp. Strain PCC 6803
Authors: Richter Stefan , Hagemann Martin , Messer Walter
Published: December 2019

Transcription of the
dnaA
gene of the cyanobacterium
Synechocystis
sp. strain PCC 6803 is light dependent and yields a monocistronic mRNA, as determined by Northern analysis. Surprisingly, mutants with inactivated
dnaA
were viable. In batch cultures under standard conditions, the mutants grew like the wild type and did not show an aberrant phenotype. We conclude that, unlike the situation in other bacteria,
dnaA
of
Synechocystis
sp. cannot have an essential function, such as initiation of DNA replication.
New Potential Cell Wall Glucanases of
Saccharomyces cerevisiae
and Their Involvement in Mating
Authors: Cappellaro Corinna , Mrsa Vladimir , Tanner Widmar
Published: December 2019

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

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

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

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

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

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

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

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

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

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

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.