bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in
Bacillus megaterium
Authors: McCool Gabriel J. , Cannon Maura C.
Published: December 2019

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in
Bacillus megaterium
11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5
pha
genes,
phaP
, -
Q
, -
R
, -
B
, and -
C
. The
phaP
and -
Q
genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream,
phaR
, -
B
, and -
C
were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to
Escherichia coli
and to a PhaC
−
mutant of
Pseudomonas putida
gave a Pha
+
phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.
S-Methylmethionine Metabolism inEscherichia coli
Authors: Thanbichler Martin , Neuhierl Bernhard , Böck August
Published: December 2019

Selenium-accumulatingAstragalusspp. contain an enzyme which specifically transfers a methyl group fromS-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed thatEscherichia colipossesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is anS-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants inyagDwhich also possess defects inmetEandmetHare unable to utilizeS-methylmethionine for growth, whereas ametE metHdouble mutant still grows onS-methylmethionine. Upstream ofyagDand overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in ametE metHgenetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter forS-methylmethionine. Methionine but notS-methylmethionine in the medium reduces the amount ofyagDprotein. This and the existence of four MET box motifs upstream ofyfkDindicate that the two genes are members of the methionine regulon. The physiological roles of theykfDandyagDproducts appear to reside in the acquisition ofS-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.
Escherichia coli
Lrp (Leucine-Responsive Regulatory Protein) Does Not Directly Regulate Expression of the
leu
Operon Promoter
Authors: Landgraf Jeffrey R. , Boxer Jonathan A. , Calvo Joseph M.
Published: December 2019

Studies by R. Lin et al. (J. Bacteriol. 174:1948–1955, 1992) suggested that the
Escherichia coli leu
operon might be a member of the Lrp regulon. Their results were obtained with a leucine auxotroph; in leucine prototrophs grown in a medium lacking leucine, there was little difference in
leu
operon expression between
lrp
+
and
lrp
strains. Furthermore, when
leuP-lacZ
transcriptional fusions that lacked the
leu
attenuator were used, expression from the
leu
promoter varied less than twofold between
lrp
+
and
lrp
strains, irrespective of whether or not excess leucine was added to the medium. The simplest explanation of the observations of Lin et al. is that the known elevated leucine transport capacity of
lrp
strains (S. A. Haney et al., J. Bacteriol. 174:108–115, 1992) leads to very high intracellular levels of leucine for strains grown with leucine, resulting in the superattenuation of
leu
operon expression.
Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in
Staphylococcus aureus
Infection
Authors: Ji Yinduo , Marra Andrea , Rosenberg Martin , Woodnutt Gary
Published: December 2019

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted a
tet
regulatory expression system for use in
Staphylococcus aureus
, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the
S. aureus
alpha-toxin gene (
hla
) in antisense orientation downstream of the
tet
promoter system and introduced the construct into
S. aureus
. Induced antisense
hla
RNA downregulated chromosomally derived
hla
gene expression in vitro approximately 14-fold. Similarly, induction of
hla
antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of
S. aureus
infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the
tet
regulatory system functions efficiently in
S. aureus
and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.
The
Oenococcus oeni clpX
Homologue Is a Heat Shock Gene Preferentially Expressed in Exponential Growth Phase
Authors: Jobin Michel-Philippe , Garmyn Dominique , Diviès Charles , Guzzo Jean
Published: December 2019

Using degenerated primers from conserved regions of previously studied
clpX
gene products, we cloned the
clpX
gene of the malolactic bacterium
Oenococcus oeni
. The
clpX
gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed
clpX
gene products from other organisms. An open reading frame located upstream of the
clpX
gene was identified as the
tig
gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic
clpX
and
tig
mRNAs and also showed an increase in
clpX
mRNA amount after a temperature shift from 30 to 42°C. The
clpX
transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike
hsp18
, the gene encoding one of the major small heat shock proteins of
Oenococcus oeni
,
clpX
expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5′ end of
clpX
mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as
Escherichia coli.
Activation of the Cryptic
aac(6′)-Iy
Aminoglycoside Resistance Gene of
Salmonella
by a Chromosomal Deletion Generating a Transcriptional Fusion
Authors: Magnet Sophie , Courvalin Patrice , Lambert Thierry
Published: December 2019

Salmonella enterica
subsp.
enterica
serotype Enteritidis BM4361 and BM4362 were isolated from the same patient. BM4361 was susceptible to aminoglycosides, whereas BM4362 was resistant to tobramycin owing to synthesis of a 6′-
N
-acetyltransferase type I [AAC(6′)-I]. Comparative analysis of nucleotide sequences, pulsed-field gel electrophoresis patterns, and Southern hybridizations indicated that the chromosomal
aac(6′)-Iy
genes for the enzyme in both strains were identical and that BM4362 derived from BM4361 following a ca. 60-kb deletion that occurred 1.5 kb upstream from the resistance gene. Northern hybridizations showed that
aac(6′)-Iy
was silent in BM4361 and highly expressed in BM4362 due to a transcriptional fusion. Primer extension mapping identified the transcriptional start site for
aac(6′)-Iy
in BM4362: 5 bp downstream from the promoter of the
nmpC
gene. Study of the distribution of
aac(6′)-Iy
by PCR and Southern hybridization with a specific probe indicated that the gene, although not found in
S. enterica
subsp.
arizonae
, was specific for
Salmonella
. In this bacterial genus,
aac(6′)-Iy
was located downstream from a cluster of seven open reading frames analogous to an
Escherichia coli
locus that encodes enzymes putatively involved in carbohydrate transport or metabolism. This genomic environment suggests a role in the catabolism of a specific sugar for AAC(6′)-Iy in
Salmonella
.
Acetate Metabolism in a
pta
Mutant of
Escherichia coli
W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival
Authors: Chang Dong-Eun , Shin Sooan , Rhee Joon-Shick , Pan Jae-Gu
Published: December 2019

In order to study the physiological role of acetate metabolism in
Escherichia coli
, the growth characteristics of an
E. coli
W3100
pta
mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The
pta
mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,
d
-lactate, and
l
-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the
pta
mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The
pta
mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the
pta
mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (
phbCAB
) from
Alcaligenes eutrophus
, indicating that the growth defect of the
pta
mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that
E. coli
excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the
pta
mutant.
Stable Packaging of Phage PRD1 DNA Requires Adsorption Protein P2, Which Binds to the IncP Plasmid-Encoded Conjugative Transfer Complex
Authors: Grahn A. Marika , Caldentey Javier , Bamford Jaana K. H. , Bamford Dennis H.
Published: December 2019

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent

K
d

of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.
Loss of Cytochrome
c
Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of
Azospirillum lipoferum
Authors: Alexandre Gladys , Bally René , Taylor Barry L. , Zhulin Igor B.
Published: December 2019

Laccase, a
p
-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium,
Azospirillum lipoferum
. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome
c
oxidase of the
aa
3
type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the
bc
1
complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome
c
oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.
Outer Membrane Lipoprotein
e
(P4) of
Haemophilus influenzae
Is a Novel Phosphomonoesterase
Authors: Reilly Thomas J. , Chance Deborah L. , Smith Arnold L.
Published: December 2019

Haemophilus influenzae
exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable
H. influenzae
strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein
e
(P4) within the
H. influenzae
sequence database.
Escherichia coli
DH5α transformed with plasmids containing the
H. influenzae hel
gene, which encodes lipoprotein
e
(P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated ∼28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase.
Deletion of the
pyc
Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit
Authors: Pérez-Redondo Rosario , Rodríguez-García Antonio , Martín Juan F. , Liras Paloma
Published: December 2019

The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (
pyc
, for pyruvate converting) located upstream of the
bls
gene in the clavulanic acid gene cluster of
Streptomyces clavuligerus
encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the
pyc
gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the
pyc
gene with the
aph
gene did not cause isoleucine-valine auxotrophy in the mutant. The
pyc
replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the
pyc
gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing
d
-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The
pyc
replacement mutant overproduces cephamycin.
A RecG-Independent Nonconservative Branch Migration Mechanism inEscherichia coliRecombination
Authors: Friedman-Ohana Rachel , Karunker Iris , Cohen Amikam
Published: December 2019

To gain insight regarding the mechanisms that extend heteroduplex joints inEscherichia colirecombination, we investigated the effect ofrecGandruvgenotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and inruvmutants. However, inrecGmutants heteroduplexes were generated almost exclusively by pairing the invasive 3′-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect ofrecGandrecJorxonAmutations on recombination proficiency and the inhibitory effect ofrecJandxonAactivities on heteroduplex formation by the 5′-ending strands are consistent with this proposal.
Direct Selection of IS
903
Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of
Actinobacillus actinomycetemcomitans
Authors: Thomson Valeri J. , Bhattacharjee Mrinal K. , Fine Daniel H. , Derbyshire Keith M. , Figurski David H.
Published: December 2019

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS
903
-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS
903φkan
, carries a cryptic
kan
gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS
903φkan
was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS
903φkan
insertion mutants were readily selected in
Escherichia coli
by their resistance to kanamycin. We used IS
903φkan
to isolate three catalase-deficient mutants of the periodontal pathogen
Actinobacillus actinomycetemcomitans
from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS
903φkan
insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated
katA
) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of
Haemophilus influenzae
. Our results demonstrate the utility of the IS
903φkan
mutagenesis system for the study of
A. actinomycetemcomitans
. Because IS
903φkan
is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
Regulation of Sigma 54-Dependent Transcription by Core Promoter Sequences: Role of −12 Region Nucleotides
Authors: Wang Lei , Guo Yuli , Gralla Jay D.
Published: December 2019

The tetranucleotide core recognition sequence (TTGC) of the sigma 54 promoter −12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo and in vitro. Deregulated promoters were identified, implying that this core element can mediate the response to enhancer-binding proteins. These promoters had in common a substitution at position −12 (consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were identified. Their analysis indicated that positions −13 (consensus G) and −15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the −13 and −15 positions contribute to closed-complex formation, whereas the −12 position has a stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that the −12 region core contains specific subsequences that direct the diverse RNA polymerase interactions required both to produce RNA and to restrict this RNA synthesis in the absence of activation.
IS
1630
of
Mycoplasma fermentans
, a Novel IS
30
-Type Insertion Element That Targets and Duplicates Inverted Repeats of Variable Length and Sequence during Insertion
Authors: Calcutt Michael J. , Lavrrar Jennifer L. , Wise Kim S.
Published: December 2019

A new insertion sequence (IS) of
Mycoplasma fermentans
is described. This element, designated IS
1630
, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS
30
family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS
30
. Multiple copies of IS
1630
are present in the genomes of at least two
M. fermentans
strains. Characterization and comparison of nine copies of the element revealed that IS
1630
exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS
1630
was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS
30
-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS
1630
vary between 19 and 26 bp. With the exception of two recently reported IS
4
-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037–1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319–1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS
1630
-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS
30
is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS
1630
appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence IS
Mi
1
was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS
3
family members than had previously been recognized. A potential site for programmed translational frameshifting in IS
Mi
1
was also identified.
Evidence that Expression of the
Vibrio vulnificus
Hemolysin Gene Is Dependent on Cyclic AMP and Cyclic AMP Receptor Protein
Authors: Bang Young Bae , Lee Shee Eun , Rhee Joon Haeng , Choi Sang Ho
Published: December 2019

Glucose repressed hemolysin production in
Vibrio vulnificus
. Promoter activity of the hemolysin gene,
vvh
, assessed with a
vvh-luxCDABE
transcriptional fusion, required cyclic AMP (cAMP) and cAMP receptor protein (CRP) in
Escherichia coli
. Hemolysin production in
V. vulnificus
increased after the addition of cAMP and was undetectable in a putative
crp
mutant, suggesting that
vvh
is also regulated by cAMP-CRP in
V. vulnificus
.
Cloning and Characterization of a Gene (
mspA
) Encoding the Major Sheath Protein of
Treponema maltophilum
ATCC 51939
T
Authors: Heuner Klaus , Choi Bong-Kyu , Schade Rüdiger , Moter Annette , Otto Albrecht , Göbel Ulf B.
Published: December 2019

The major sheath protein-encoding gene (
mspA
) of the oral spirochete
Treponema maltophilum
ATCC 51939
T
was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The
mspA
gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an
mspA
-specific probe showed no cross-reactivity with the
msp
gene from
Treponema denticola
.
Molecular Characterization of the
Lactococcus lactis ptsHI
Operon and Analysis of the Regulatory Role of HPr
Authors: Luesink Evert J. , Beumer Christel M. A. , Kuipers Oscar P. , De Vos Willem M.
Published: December 2019

The
Lactococcus lactis ptsH
and
ptsI
genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the
ptsHI
operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing
ptsH
and a second of 2.0 kb containing both
ptsH
and
ptsI
. Disruption of the
ptsH
and
ptsI
genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in
L. lactis
. Complementation of the
ptsH
and
ptsI
mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the
gal
operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and
l
-lactate dehydrogenase genes.
Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability
Authors: Samartzidou Hrissi , Delcour Anne H.
Published: December 2019

The permeability of the outer membrane of
Escherichia coli
to hydrophilic compounds is controlled by porin channels. Electrophysiological experiments showed that polyamines inhibit ionic flux through cationic porins when applied to either side of the membrane. Externally added polyamines, such as cadaverine, decrease porin-mediated fluxes of β-lactam antibiotics in live cells. Here we tested the effects of endogenously expressed cadaverine on the rate of permeation of cephaloridine through porins, by manipulating in a pH-independent way the expression of the
cadBA
operon, which encodes proteins involved in the decarboxylation of lysine to cadaverine and in cadaverine excretion. We report that increased levels of excreted cadaverine correlate with a decreased outer membrane permeability to cephaloridine, without any change in porin expression. Cadaverine appears to promote a sustained inhibition of porins, since the effect remains even after removal of the exogenously added or excreted polyamine. The cadaverine-induced inhibition is sufficient to provide cells with some resistance to ampicillin but not to hydrophobic antibiotics. Finally, the mere expression of
cadC
, in the absence of cadaverine production, leads to a reduction in the amounts of OmpF and OmpC proteins, which suggests a novel mechanism for the environmental control of porin expression. The results presented here support the notion that polyamines can act as endogenous modulators of outer membrane permeability, possibly as part of an adaptive response to acidic conditions.
Immunochemical Analysis of UMP Kinase from
Escherichia coli
Authors: Landais Stéphanie , Gounon Pierre , Laurent-Winter Christine , Mazié Jean-Claude , Danchin Antoine , Bârzu Octavian , Sakamoto Hiroshi
Published: December 2019

Mono- and polyclonal antibodies directed against UMP kinase from
Escherichia coli
were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole
E. coli
cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.
PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of
Pseudomonas oleovorans
GPo1 Involved in the Regulatory Expression System for
pha
Genes
Authors: Prieto Maria A. , Bühler Bruno , Jung Kuno , Witholt Bernard , Kessler Birgit
Published: December 2019

The
phaC1
gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of
Pseudomonas oleovorans
GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant
P. oleovorans
strain carrying a
phaC1
::
lacZ
reporter system, that the
phaC1
gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a
P. oleovorans
GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn
5
insertional mutagenesis. Characterization of this mutant allowed us to conclude that
phaF
, a gene located downstream of the
pha
gene cluster, was knocked out in this strain.
P. oleovorans
GPG-Tc6 regained the ability to control
phaC1
gene expression when complemented with the
phaF
wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and
phaI
and
phaF
genes. The amino acid sequences of the
phaI
gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing
phaF
, i.e.,
phaF
and
phaIF
transcripts. Expression of the
phaIF
operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by
P. oleovorans
. A model for the role of PhaF in regulating PHA synthesis is presented.
NolL of
Rhizobium
sp. Strain NGR234 Is Required for
O
-Acetyltransferase Activity
Authors: Berck S. , Perret X. , Quesada-Vincens D. , Promé J.-C. , Broughton W. J. , Jabbouri S.
Published: December 2019

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium
Rhizobium
sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible
nolL
gene, which is preceded by a
nod
box, results in the synthesis of NodNGR factors that lack the 3-
O
- or 4-
O
-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩ
nolL
is not impaired, whereas introduction of the
nod
box::
nolL
construct into the related strain
Rhizobium fredii
USDA257 extends the host range of this bacterium to
Calopogonium caeruleum
,
Leucaena leucocephala
, and
Lotus halophilus
. Nod factors produced by a USDA257(p
nolL
) transconjugant were also acetylated. The
nod
box::
nolL
construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the
nodD1
gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.
Hydride-Meisenheimer Complex Formation and Protonation as Key Reactions of 2,4,6-Trinitrophenol Biodegradation by
Rhodococcus erythropolis
Authors: Rieger Paul-Gerhard , Sinnwell Volker , Preuß Andrea , Francke Wittko , Knackmuss Hans-Joachim
Published: December 2019

Biodegradation of 2,4,6-trinitrophenol (picric acid) by
Rhodococcus erythropolis
HLPM-1 proceeds via initial hydrogenation of the aromatic ring system. Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic ς-complex) of picric acid and its protonated form under physiological conditions. These complexes are key intermediates of denitration and productive microbial degradation of picric acid. For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time. Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid. This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite. Cell extracts of
R. erythropolis
HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol. Picric acid is used as the sole carbon, nitrogen, and energy source by
R. erythropolis
HLPM-1.
Characterization of Insertions of IS
476
and Two Newly Identified Insertion Sequences, IS
1478
and IS
1479
, in
Xanthomonas campestris
pv. campestris
Authors: Chen Jiann-Hwa , Hsieh Yu-Ying , Hsiau Su-Lian , Lo Ta-Chun , Shau Chen-Chun
Published: December 2019

Thirty-two plasmid insertion mutants were independently isolated from two strains of
Xanthomonas campestris
pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS
3
family member, IS
476
, and two new insertion sequences (IS), IS
1478
and IS
1479
. While IS
1478
does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS
1479
demonstrated 73% sequence homology with IS
1051
in
X. campestris
pv. dieffenbachiae, 62% homology with IS
52
in
Pseudomonas syringae
pv. glycinea, and 60% homology with IS
5
in
Escherichia coli
. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS
1478
by itself constitutes a new subfamily of the widespread IS
5
family, whereas IS
1479
, along with IS
1051
, IS
52
, and IS
5
, belongs to the IS
5
subfamily of the IS
5
family. All but one of the IS
476
insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS
476
insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS
1478
and IS
1479
, on the other hand, were not random, and IS
1478
and IS
1479
each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS
476
, IS
1478
, and IS
1479
are present in the genomes of all seven
X. campestris
pv. campestris strains tested and several
X. campestris
pathovars.
Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase fromPseudomonas aeruginosa
Authors: Velasco-García Roberto , Mújica-Jiménez Carlos , Mendoza-Hernández Guillermo , Muñoz-Clares Rosario A.
Published: December 2019

Betaine aldehyde dehydrogenase (BADH) (EC1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. InPseudomonas aeruginosathis reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2′,5′-ADP–Sepharose, which results in a high yield of pure enzyme with a specific activity at 30°C and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH fromP. aeruginosais a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes fromXanthomonas translucensandEscherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, thePseudomonasenzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol−1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 ± 0.1 and 9.7 ± 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40°C, but activity is regained when the heated enzyme is cooled to 30°C or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also,P. aeruginosaBADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+considerably protected the enzyme against these inactivating conditions.
IS
1634
, a Novel Insertion Element Creating Long, Variable-Length Direct Repeats Which Is Specific for
Mycoplasma mycoides
subsp.
mycoides
Small-Colony Type
Authors: Vilei Edy M. , Nicolet Jacques , Frey Joachim
Published: December 2019

A new insertion sequence, IS
1634
, has been identified in
Mycoplasma mycoides
subsp.
mycoides
small-colony type (SC). IS
1634
shows structural and functional similarities to IS
1549
of
Mycobacterium smegmatis
and with it seems to form a new class or family of insertion sequences. IS
1634
has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS
1549
and to a lesser extent to the transposases of IS elements of the IS
4
family. IS
1634
is present at about 30 copies in the genome of all 22 different field strains of
M. mycoides
subsp.
mycoides
SC tested. Characteristic of IS
1634
are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS
1634
is specific to
M. mycoides
subsp.
mycoides
SC and is not present in any of the other members of the
M. mycoides
cluster. Neither was it found in other closely related
Mycoplasma
species of ruminants.
Natural Competence for DNA Transformation by
Legionella pneumophila
and Its Association with Expression of Type IV Pili
Authors: Stone Barbara J. , Kwaik Yousef Abu
Published: December 2019

We have recently described the expression of two pili of different lengths on the surface of
Legionella pneumophila
(B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768–1775, 1998). Production of long pili requires a functional
pilE
L
locus, encoding a type IV pilin protein. Since type IV pili in
Neisseria gonorrhoeae
are associated with competence for DNA transformation, we examined the competence of
L. pneumophila
for DNA transformation under conditions that allowed the expression of type IV pili. We show that
L. pneumophila
is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing
L. pneumophila
DNA. Many different
L. pneumophila
loci are able to transform
L. pneumophila
after addition of plasmid DNA, including
gspA
,
ppa
,
asd
, and
pilE
L
. The transformation frequency is reduced when competing DNA containing either
L. pneumophila
DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a
pilE
L
mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of
pilE
L
alone. We conclude that competence for DNA transformation in
L. pneumophila
is associated with expression of the type IV pilus and results in recombination of
L. pneumophila
DNA into the chromosome. Since expression of type IV pili also facilitates attachment of
L. pneumophila
to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).
Mutational Analysis of
Bacillus subtilis
Glutamine Phosphoribosylpyrophosphate Amidotransferase Propeptide Processing
Authors: Li Songtao , Smith Janet L. , Zalkin Howard
Published: December 2019

Glutamine phosphoribosylpyrophosphate amidotransferase from
Bacillus subtilis
is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.
Cloning and Expression of the
algL
Gene, Encoding the
Azotobacter chroococcum
Alginate Lyase: Purification and Characterization of the Enzyme
Authors: Peciña Ana , Pascual Alberto , Paneque Antonio
Published: December 2019

The alginate lyase-encoding gene (
algL
) of
Azotobacter chroococcum
was localized to a 3.1-kb
Eco
RI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the
Azotobacter vinelandii
gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the
Pseudomonas aeruginosa
gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The
A. chroococcum
AlgL protein was overproduced in
Escherichia coli
and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.
Inhibition of Translation and Cell Growth by Minigene Expression
Authors: Tenson Tanel , Herrera Jesús Vega , Kloss Patricia , Guarneros Gabriel , Mankin Alexander S.
Published: December 2019

A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.
A Cold Shock-Induced Cyanobacterial RNA Helicase
Authors: Chamot Danuta , Magee Wendy C. , Yu Esther , Owttrim George W.
Published: December 2019

The ability to modify RNA secondary structure is crucial for numerous cellular processes. We have characterized two RNA helicase genes,
crhB
and
crhC
, which are differentially expressed in the cyanobacterium
Anabaena
sp. strain PCC 7120.
crhC
transcription is limited specifically to cold shock conditions while
crhB
is expressed under a variety of conditions, including enhanced expression in the cold. This implies that both RNA helicases are involved in the cold acclimation process in cyanobacteria; however, they presumably perform different roles in this adaptation. Although both CrhB and CrhC belong to the DEAD box subfamily of RNA helicases, CrhC encodes a novel RNA helicase, as the highly conserved SAT motif is modified to FAT. This alteration may affect CrhC function and its association with specific RNA targets and/or accessory proteins, interactions required for cold acclimation. Primer extension and analysis of the 5′ untranslated region of
crhC
revealed the transcriptional start site, as well as a number of putative cold shock-responsive elements. The potential role(s) performed by RNA helicases in the acclimation of cyanobacteria to cold shock is discussed.