bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of
dacC
, Which Encodes a New Low-Molecular-Weight Penicillin-Binding Protein in
Bacillus subtilis
Authors: Pedersen Lotte B. , Murray Thomas , Popham David L. , Setlow Peter
Published: December 2019

The
pbp
gene (renamed
dacC
), identified by the
Bacillus subtilis
genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional
dacC-lacZ
fusion revealed that
dacC
expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor ς
H
; and (iii) appears to be initiated from a promoter located immediately upstream of
yoxA
, a gene of unknown function located upstream of
dacC
on the
B. subtilis
chromosome. A
B. subtilis dacC
insertional mutant grew and sporulated identically to wild-type cells, and
dacC
and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics. Expression of
dacC
in
Escherichia coli
showed that this gene encodes an ∼59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.
Heterologous Expression of the
Desulfovibrio gigas
[NiFe] Hydrogenase in
Desulfovibrio fructosovorans
MR400
Authors: Rousset Marc , Magro Valérie , Forget Nicole , Guigliarelli Bruno , Belaich Jean-Pierre , Hatchikian E. Claude
Published: December 2019

The ability of
Desulfovibrio fructosovorans
MR400 Δ
hynABC
to express the heterologous cloned [NiFe] hydrogenase of
Desulfovibrio gigas
was investigated. The [NiFe] hydrogenase operon from
D. gigas
,
hynABCD
, was cloned, sequenced, and introduced into
D. fructosovorans
MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native
D. gigas
protein. A chimeric operon containing
hynAB
from
D. gigas
and
hynC
from
D. fructosovorans
placed under the control of the
D. fructosovorans hynAp
promoter was constructed and expressed in
D. fructosovorans
MR400. Under these conditions, the same level of activity was obtained as with the
D. gigas
hydrogenase operon.
A New Class of
Caulobacter crescentus
Flagellar Genes
Authors: Leclerc Guy , Wang Shui Ping , Ely Bert
Published: December 2019

Eight
Caulobacter crescentus
flagellar genes,
flmA
,
flmB
,
flmC
,
flmD
,
flmE
,
flmF
,
flmG
, and
flmH
, have been cloned and characterized. These eight genes are clustered in pairs (
flmAB
,
flmCD
,
flmEF
, and
flmGH
) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the
flm
genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the
flmAB
,
flmEF
, and
flmGH
operons was shown to occur primarily in predivisional cells. In contrast, the
flmCD
operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the
flm
genes. Furthermore, the
flm
genes did not affect the expression of class II or class III flagellar genes. However, mutations in the
flm
genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the
flm
operons belong to a new class of flagellar genes.
Rare Homologous Gene Targeting in
Histoplasma capsulatum
: Disruption of the
URA5
Hc
Gene by Allelic Replacement
Authors: Woods Jon P. , Retallack Diane M. , Heinecke Elizabeth L. , Goldman William E.
Published: December 2019

URA5
genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the
Histoplasma capsulatum URA5
gene (
URA5
Hc
) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized
ura5
strains of
H. capsulatum
. We attempted to target the genomic
URA5
locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in
H. capsulatum
and has been challenging in some other pathogenic fungi. Several strategies commonly used in
Saccharomyces cerevisiae
and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for
URA5
gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (
hph
, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Δ
ura5
Hc
::
hph
plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in
H. capsulatum
, or in any dimorphic systemic fungal pathogen of humans.
A Complex Control System for Transcriptional Activation from the
sid
Promoter of Bacteriophage P4
Authors: Reiter Kaye , Lam Hugh , Young Edward , Julien Bryan , Calendar Richard
Published: December 2019

The
sid
gene promoter (P

sid

), which controls expression of the late genes from satellite phage P4, is activated by a unique class of small DNA-binding proteins. The activators from both satellite and helper phages stimulate transcription from P

sid

. These activators bind to sites centered at position −55 in all the helper and satellite phage late promoters. P4 P

sid

is unique in that it has an additional activator binding site centered at position −18 (site II). We have constructed a mutant of site II that no longer binds activators. Transcription under the control of satellite phage activators is increased by the site II mutation. In contrast, helper phage activators do not show this increase in transcription from P

sid

mutated at site II. Competition gel shift analysis reveals that the P4 satellite phage activator, Delta, binds eightfold better to site II than to site I. The products of the
sid
transcription unit are needed only when a helper phage is present; thus, the satellite phage activators repress transcription until the helper is present to supply a nonrepressing activator.
Bradyrhizobium japonicum
FixK
2
, a Crucial Distributor in the FixLJ-Dependent Regulatory Cascade for Control of Genes Inducible by Low Oxygen Levels
Authors: Nellen-Anthamatten D. , Rossi P. , Preisig O. , Kullik I. , Babst M. , Fischer H. M. , Hennecke H.
Published: December 2019

Bradyrhizobium japonicum
possesses a second
fixK
-like gene,
fixK
2
, in addition to the previously identified
fixK
1
gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates
fixK
2
, whose product then activates
fixK
1
. While the target genes for control by FixK
1
are unknown, there is evidence for activation of the
fixNOQP
,
fixGHIS
, and
rpoN
1
genes and some heme biosynthesis and nitrate respiration genes by FixK
2
. FixK
2
also regulates its own structural gene, directly or indirectly, in a negative way.
Extracellular Signal Molecule(s) Involved in the Carbon Starvation Response of Marine
Vibrio
sp. Strain S14
Authors: Srinivasan Sujatha , Östling Jörgen , Charlton Timothy , de Nys Rocky , Takayama Kathy , Kjelleberg Staffan
Published: December 2019

The role of exogenous metabolites as putative signal molecules mediating and/or regulating the carbon starvation adaptation program in
Vibrio
sp. strain S14 was investigated. Addition of the stationary-phase supernatant extract (SSE) of
Vibrio
sp. strain S14 to logarithmic-phase cells resulted in a significant number of carbon starvation-induced proteins being up-regulated. Halogenated furanones, putative antagonists of acylated homoserine lactones (AHLs), inhibited the synthesis of proteins specifically induced upon carbon starvation. The effect of the furanone was the opposite of that caused by SSE with respect to the up- and down-regulation of protein expression, indicating that both the furanone and the putative signalling molecules were acting on the same regulatory pathway. Culturability was rapidly lost when
Vibrio
sp. strain S14 was starved in the presence of the furanone at a low concentration. The furanone also had a negative effect on the ability of carbon-starved cells to mount resistance against UV irradiation and hydrogen peroxide exposure. The SSE of
Vibrio
sp. strain S14 had the ability to provide cross-protection against the loss in viability caused by the furanone. We have further demonstrated that the SSE taken from low- as well as high-cell-density cultures of
Vibrio
sp. strain S14 induced luminescence in
Vibrio harveyi
. Taken together, the results in this report provide evidence that
Vibrio
sp. strain S14 produces extracellular signalling metabolites during carbon and energy starvation and that these molecules play an important role in the expression of proteins crucial to the development of starvation- and stress-resistant phenotypes.
Cell Reproduction and Morphological Changes in
Mycoplasma capricolum
Authors: Seto Shintaro , Miyata Makoto
Published: December 2019

The cell reproduction of
Mycoplasma capricolum
was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of
Escherichia coli
. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0.
M. capricolum
cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.
Initiation and Velocity of Chromosome Replication in
Escherichia coli
B/r and K-12
Authors: Bipatnath Meeshel , Dennis Patrick P. , Bremer Hans
Published: December 2019

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common
Escherichia coli
strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (
C
period) were determined. For these two strains, the initiation mass approached values of 8 × 10
−10
and 10 × 10
−10
units of optical density (at 460 nm) of culture mass per
oriC
, respectively, at growth rates above 1 doubling/h (at 37°C). The amount of protein per
oriC
decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The
C
period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and
C
period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.
Properties of the P-Type ATPases Encoded by the
copAP
Operons of
Helicobacter pylori
and
Helicobacter felis
Authors: Bayle Denis , Wängler Sabine , Weitzenegger Thomas , Steinhilber Wolfram , Volz Jürgen , Przybylski Michael , Schäfer Klaus P. , Sachs George , Melchers Klaus
Published: December 2019

The
cop
operons of
Helicobacter pylori
and
Helicobacter felis
were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd
2+
and Cu
2+
ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the
copYZAB
operon of
Enterococcus hirae
. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the
H. pylori
CopA ATPase bound to Cu
2+
specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of
H. pylori
to Cu
2+
but not to other divalent cations. As determined experimentally,
H. pylori
CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of
H. pylori
(K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the
H. felis
CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu
2+
binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that
H. pylori
and
H. felis
employ conserved mechanisms of ATPase-dependent copper resistance.
MinCD Proteins Control the Septation Process during Sporulation of
Bacillus subtilis
Authors: Barák Imrich , Prepiak Peter , Schmeisser Falko
Published: December 2019

Mutation of the
divIVB
locus in
Bacillus subtilis
causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The
divIVB
locus contains five open reading frames (ORFs). The last two ORFs (
minCD
) are homologous to
minC
and
minD
of
Escherichia coli
but a
minE
homolog is lacking in
B. subtilis
. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. However, it has been proposed that MinCD has no essential role in sporulation septum formation. We have used electron microscopic studies to show septation events during sporulation in some
minD
strains. We have observed an unusually thin septum at the midcell position in
minD
and also in
minD spoIIE71
mutant cells. Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site in
minD
cells. We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation of
B. subtilis
cells.
Escherichia coli
Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters
Authors: Ross Wilma , Aiyar Sarah E. , Salomon Julia , Gourse Richard L.
Published: December 2019

The α subunit of
Escherichia coli
RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter
rrnB
P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional
E. coli
promoters (
rrnD
P1,
rrnB
P2, λ
p
R
,
lac
,
merT
, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the
lac
core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.
Redox-Dependent Gene Regulation in
Rhodobacter sphaeroides
2.4.1
T
: Effects on Dimethyl Sulfoxide Reductase (
dor
) Gene Expression
Authors: Mouncey Nigel J. , Kaplan Samuel
Published: December 2019

The ability of
Rhodobacter sphaeroides
2.4.1
T
to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine
N
-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the
dor
locus. Previously, we have demonstrated that
dor
expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of
dor
expression, we measured DMSO reductase synthesis and β-galactosidase activity from
dor
::
lacZ
fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically,
dorC
::
lacZ
expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the
ccoP
mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered
dor
expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in
R. sphaeroides
and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling
dor
expression.
Identification of an N-Terminal Region of Sigma 54 Required for Enhancer Responsiveness
Authors: Syed Adeela , Gralla Jay D.
Published: December 2019

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.
The Phenolic Recognition Profiles of the
Agrobacterium tumefaciens
VirA Protein Are Broadened by a High Level of the Sugar Binding Protein ChvE
Authors: Peng Wen-Tao , Lee Yong-Woog , Nester Eugene W.
Published: December 2019

The formation of crown gall tumors by
Agrobacterium tumefaciens
requires that the virulence (
vir
) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites. Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding protein which interacts with VirA. We have previously presented genetic evidence that
virA
senses phenolic compounds directly (Y.-W. Lee, S. Jin, W.-S. Sim, and E. W. Nester, Proc. Natl. Acad. Sci. USA 92:12245–12249, 1995). The
vir
genes of strain KU12 can be induced by 4-hydroxyacetophenone,
p
-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the
vir
genes of strain A6. In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses. 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the
virA
gene of A6 if arabinose replaces glucose as the inducing sugar. We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory protein GbpR. If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the
vir
genes to high levels in combination with glucose. Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain. We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.
Induction of
ermAMR
from a Clinical Strain of
Enterococcus faecalis
by 16-Membered-Ring Macrolide Antibiotics
Authors: Oh Tae-Gwon , Kwon Ae-Ran , Choi Eung-Chil
Published: December 2019

We cloned the MLS
B
resistance determinant by PCR from a clinical isolate of
Enterococcus faecalis
373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of
E. faecalis
373, we analyzed the cloned gene, designated
ermAMR
, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.
Identification of a Zinc-Specific Metalloregulatory Protein, Zur, Controlling Zinc Transport Operons in
Bacillus subtilis
Authors: Gaballa Ahmed , Helmann John D.
Published: December 2019

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in
Bacillus subtilis
. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake,
yciC
and
ycdHIyceA
. A
zur
mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the
yciC
control region. A similar operator precedes the
ycdH
-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the
ycdH
operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a
ycdH
mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a
yciC
mutation. Second, mutation of
ycdH
, but not
yciC
, alters the regulation of both the
yciC
and
ycdH
operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.
Targeted Mutagenesis of ς
54
Activator Proteins in
Myxococcus xanthus
Authors: Gorski Lisa , Kaiser Dale
Published: December 2019

Myxococcus xanthus
DNA segments related to the highly conserved central sequence of ς
54
activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal.
Characterization of the Lysogenic Bacteriophage MAV1 from
Mycoplasma arthritidis
Authors: Voelker LeRoy L. , Dybvig Kevin
Published: December 2019

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of
Mycoplasma arthritidis
, was characterized. Several strains of
M. arthritidis
were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (
attP
) and bacterial DNA (
attB
) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the
attB
sequence. A model for MAV1 integration-excision is proposed.
Corynebacterium glutamicum
Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP
Authors: Peter Heidi , Weil Brita , Burkovski Andreas , Krämer Reinhard , Morbach Susanne
Published: December 2019

Gram-positive soil bacterium
Corynebacterium glutamicum
uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in
C. glutamicum
, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A Δ
betP ΔputP ΔproP ΔectP
mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (

K
m

, 48 μM) to ectoine (

K
m

, 132 μM). The
proP
gene could not be expressed from its own promoter in
C. glutamicum
; however, expression was observed in
Escherichia coli
. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed
ectP
codes for a 615-residue transporter. EctP preferred ectoine (

K
m

, 63 μM) to betaine (

K
m

, 333 μM) and proline (

K
m

, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.
Transport of Intact Porphyrin by HpuAB, the Hemoglobin-Haptoglobin Utilization System ofNeisseria meningitidis
Authors: Lewis L. A. , Sung M.-H. , Gipson M. , Hartman K. , Dyer D. W.
Published: December 2019

The meningococcalhemAgene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of thehemAmutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA−HpuAB−double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.
An Upstream Sequence Element Required for NucC-Dependent Expression of the
Serratia marcescens
Extracellular Nuclease
Authors: Winslow Robert H. , Julien Bryan , Calendar Richard , Christie Gail E.
Published: December 2019

The
Serratia marcescens
extracellular nuclease gene,
nucA
, is positively regulated by the product of the
nucC
gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (
cat
). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between −76 and −59 relative to the start of transcription. The activator binding site in the
nucA
promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative −35 region are consistent with a strict helical phasing requirement for activation.
Sequence Diversity of the
oprI
Gene, Coding for Major Outer Membrane Lipoprotein I, among rRNA Group I Pseudomonads
Authors: De Vos Daniel , Bouton Christiane , Sarniguet Alain , De Vos Paul , Vauterin Marc , Cornelis Pierre
Published: December 2019

The sequence of
oprI
, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on
Pseudomonas aeruginosa
and
Pseudomonas fluorescens
. Within the
P. aeruginosa
species,
oprI
sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The
oprI
sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with
P. aeruginosa oprI
). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the
Hae
III,
Pvu
II, and
Sph
I sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of
P. fluorescens
isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the
oprI
gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.
Gene Organization and Transcription Analysis of the
Agrobacterium tumefaciens
Glycogen (
glg
) Operon: Two Transcripts for the Single Phosphoglucomutase Gene
Authors: Ugalde Juan E. , Lepek Viviana , Uttaro Antonio , Estrella Julia , Iglesias Alberto , Ugalde Rodolfo A.
Published: December 2019

The gene organization and transcription of the
Agrobacterium glg
operon differ from those in other bacteria.
Agrobacterium tumefaciens
A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (
glgC
), glycogen synthetase (
glgA
), and phosphoglucomutase (
pgm
) genes have been previously described (A. Uttaro and R. A. Ugalde, Gene 150:117–122, 1994). In this work we report that the glycogen phosphorylase (
glgP
) and branching enzyme (
glgB
) genes are located immediately upstream of this region. The complete nucleotide sequences of the
glgP
and
glgB
genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with the wild type and with
glgP
and
glgB
mutants, as well as primer extension experiments and β-galactosidase fusions, revealed that this region containing five open reading frames (
glgPBCA
and
pgm
) is transcribed unidirectionally as a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene (
glgP
). An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of the
pgm
gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a
pgm
mutant. This alternative transcript has a promoter with the motif TATCAAN
5
G, identified in octopine Ti plasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in
A. tumefaciens
than in
Escherichia coli
, as judged by the level of enzymatic activity of a
lacZ-pgm
fusion.
Stability of the
Agrobacterium tumefaciens
VirB10 Protein Is Modulated by Growth Temperature and Periplasmic Osmoadaption
Authors: Banta Lois M. , Bohne Jutta , Lovejoy S. Dawn , Dostal Kathleen
Published: December 2019

Export of oncogenic T-DNA from the phytopathogen
Agrobacterium tumefaciens
is mediated by the products of the
virB
operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498–1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (
chvB
) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a
chvB
mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.
The Two-Component Regulators GacS and GacA Influence Accumulation of the Stationary-Phase Sigma Factor ς
S
and the Stress Response in
Pseudomonas fluorescens
Pf-5
Authors: Whistler Cheryl A. , Corbell Nathan A. , Sarniguet Alain , Ream Walter , Loper Joyce E.
Published: December 2019

Three global regulators are known to control antibiotic production by
Pseudomonas fluorescens
. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in
rpoS
, which encodes the stationary-phase sigma factor ς
S
, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of
P. fluorescens
to survive exposure to oxidative stress. The
gacA
gene of
P. fluorescens
Pf-5 was isolated, and the influence of
gacS
and
gacA
on
rpoS
transcription, ς
S
levels, and oxidative stress response of Pf-5 was determined. We selected a
gacA
mutant of Pf-5 that contained a single nucleotide substitution within a predicted α-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, ς
S
content in
gacS
and
gacA
mutants of Pf-5 was less than 20% of the wild-type level. Transcription of
rpoS
, assessed with an
rpoS-lacZ
transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in
gacS
and
gacA
compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in
P. fluorescens
.
Genome Size, Complexity, and Ploidy of the Pathogenic Fungus
Histoplasma capsulatum
Authors: Carr Jeanne , Shearer Glenmore
Published: December 2019

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus
Histoplasma capsulatum
was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, α-tubulin, and β-tubulin). G186AS was found to have a genome of approximately 2.3 × 10
7
bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 × 10
7
bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1
n
genome estimate to yield a DNA index (DNA per cell/1
n
genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with α- and β-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.
Role of the
gerI
Operon of
Bacillus cereus
569 in the Response of Spores to Germinants
Authors: Clements Mark O. , Moir Anne
Published: December 2019

Bacillus cereus
569 (ATCC 10876) germinates in response to inosine or to
l
-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to
l
-alanine were isolated after Tn 917 -LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to
l
-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to
l
-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in
l
-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of
l
-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the
gerA
and related operons of
Bacillus subtilis
. The individual genes of this
gerI
operon have been named
gerIA
,
gerIB
, and
gerIC
. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.
A Novel Serine/Threonine Protein Kinase Homologue of
Pseudomonas aeruginosa
Is Specifically Inducible within the Host Infection Site and Is Required for Full Virulence in Neutropenic Mice
Authors: Wang Jingyi , Li Caihe , Yang Hongjiang , Mushegian Arcady , Jin Shouguang
Published: December 2019

A genetic locus of
Pseudomonas aeruginosa
was identified that is highly and specifically inducible during infection of neutropenic mice. This locus,
ppkA
, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A
ppkA
null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of
Escherichia coli
or
P. aeruginosa
. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.
Active Efflux of Organic Solvents by
Pseudomonas putida
S12 Is Induced by Solvents
Authors: Kieboom Jasper , Dennis Jonathan J. , Zylstra Gerben J. , de Bont Jan A. M.
Published: December 2019

Induction of the membrane-associated organic solvent efflux system SrpABC of
Pseudomonas putida
S12 was examined by cloning a 312-bp DNA fragment, containing the
srp
promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the
srpABC
genes include aromatic and aliphatic solvents and alcohols. General stress conditions such as pH, temperature, NaCl, or the presence of organic acids did not induce
srp
transcription. Although the solvent efflux pump in
P. putida
S12 is a member of the resistance-nodulation-cell division family of transporters, the
srpABC
genes were not induced by antibiotics or heavy metals.
Identification of Two Binding Domains, One for Peptidoglycan and Another for a Secondary Cell Wall Polymer, on the N-Terminal Part of the S-Layer Protein SbsB from
Bacillus stearothermophilus
PV72/p2
Authors: Sára Margit , Egelseer Eva M. , Dekitsch Christine , Sleytr Uwe B.
Published: December 2019

First studies on the structure-function relationship of the S-layer protein from
B. stearothermophilus
PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.
Two Roles for the DNA Recognition Site of the
Klebsiella aerogenes
Nitrogen Assimilation Control Protein
Authors: Pomposiello Pablo J. , Janes Brian K. , Bender Robert A.
Published: December 2019

The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (
hutUH
) of
Klebsiella aerogenes
, and NAC bound at this site activates transcription of
hutUH
. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from
hutUH
, the NAC-binding site from the
gdhA
promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from
gdhA
allowed this site to activate transcription. Similar studies using the NAC-binding site from
hut
showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.