bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection against 3′-to-5′ RNA Decay in
Bacillus subtilis
Authors: Farr Glen A. , Oussenko Irina A. , Bechhofer David H.
Published: December 2019

A 320-nucleotide RNA with several characteristic features was expressed in
Bacillus subtilis
to study RNA processing. The RNA consisted of a 5′-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3′-proximal end of the
ermC
transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the
pnpA
gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the
pnpA
strain, suggesting that it was specific for PNPase. An abundant RNA with a 3′ end located in the
ermC
coding sequence was detected in the
pnpA
strain but not in the wild type, indicating that this block is specific for a different 3′-to-5′ exonuclease. The finding of impediments to 3′-to-5′ degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
Sliding Motility in Mycobacteria
Authors: Martínez Asunción , Torello Sandra , Kolter Roberto
Published: December 2019

Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here that
Mycobacterium smegmatis
can spread on the surface of growth medium by a sliding mechanism.
M. smegmatis
spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to
M. smegmatis
but is also a property of the slow-growing opportunistic pathogen
M. avium
. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host.
Cellobiose-6-Phosphate Hydrolase (CelF) of
Escherichia coli
: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases
Authors: Thompson John , Ruvinov Sergei B. , Freedberg Darón I. , Hall Barry G.
Published: December 2019

The gene
celF
of the cryptic
cel
operon of
Escherichia coli
has been cloned, and the encoded 6-phospho-β-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC
3.2.1.86
]) has been expressed and purified in a catalytically active state. Among phospho-β-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD
+
for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-β-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-β-glucoside-6P, and the chromogenic analog,
p
-nitrophenyl-β-
d
-glucopyranoside-6P. In the absence of a metal ion and NAD
+
, purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD
+
appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (
M
w
, ∼200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (
M
r
, ∼50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues;
M
r
= 50,512). Comparative sequence alignments provide tentative identification of the NAD
+
-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu
112
and Glu
356
) of CelF.
The Basis of Ammonium Release in
nifL
Mutants of
Azotobacter vinelandii
Authors: Brewin Brett , Woodley Paul , Drummond Martin
Published: December 2019

In
Azotobacter vinelandii
, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by
nifLA
. Certain mutations in
nifL
result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of
nif
gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination of
nifL
or overexpression of
nifA
, resulted in ammonium release, correlating with enhanced levels of
nifH
mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH
4
+
uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.
Chromate Efflux by Means of the ChrA Chromate Resistance Protein from
Pseudomonas aeruginosa
Authors: Alvarez Angel H. , Moreno-Sánchez Rafael , Cervantes Carlos
Published: December 2019

Everted membrane vesicles of
Pseudomonas aeruginosa
PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more
51
CrO
4
2−
than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent

K
m

of 0.12 mM chromate and a
V
max
of 0.5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.
Gene Disruption through Homologous Recombination in
Spiroplasma citri
: an
scm1
-Disrupted Motility Mutant Is Pathogenic
Authors: Duret Sybille , Danet Jean-Luc , Garnier Monique , Renaudin Joël
Published: December 2019

To determine whether homologous recombination could be used to inactivate selected genes in
Spiroplasma citri
, plasmid constructs were designed to disrupt the motility gene
scm1
. An internal
scm1
gene fragment was inserted into plasmid pKT1, which replicates in
Escherichia coli
but not in
S. citri
, and into the
S. citri oriC
plasmid pBOT1, which replicates in spiroplasma cells as well as in
E. coli
. Electrotransformation of
S. citri
with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the
oriC
region or at the
scm1
gene. In the latter case, plasmid integration by a single crossover between the
scm1
gene fragment carried by the plasmid and the full-length
scm1
gene carried by the chromosome led to a nonmotile phenotype. Transmission of the
scm1
-disrupted mutant to periwinkle (
Catharanthus roseus
) plants through injection into the leafhopper vector (
Circulifer haematoceps
) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type
S. citri
strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the
S. citri oriC
plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in
S. citri
, a mollicute which probably lacks a functional RecA protein.
A Cyclic AMP Receptor Protein Mutant That Constitutively Activates an
Escherichia coli
Promoter Disrupted by an IS
5
Insertion
Authors: Podolny Vladimir , Lin E. C. C. , Hochschild Ann
Published: December 2019

Previously an
Escherichia coli
mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the
fucO
gene (in the
fucAO
operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS
5
insertion in the intergenic regulatory region between the divergently oriented
fucAO
and
fucPIK
operons. Though expression of the
fucAO
operon was constitutive, the
fucPIK
operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the
crp
gene was selected. Here we identify this
crp
mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS
5
-disrupted and the wild-type
fucPIK
promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the
fucPIK
promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS
5
-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.
Type 1 Fimbriation and Phase Switching in a Natural
Escherichia coli fimB
Null Strain, Nissle 1917
Authors: Stentebjerg-Olesen Bodil , Chakraborty Trinad , Klemm Per
Published: December 2019

Escherichia coli
Nissle 1917 has been used as a probiotic against intestinal disorders for many decades. It is a good colonizer of the human gut and has been reported to be able to express type 1 fimbriae. Type 1 fimbriae are surface organelles which mediate α-
d
-mannose-sensitive binding to various host cell surfaces. The expression is phase variable, and two tyrosine recombinases, FimB and FimE, mediate the inversion of the fimbrial phase switch. Current evidence suggests that FimB can carry out recombination in both directions, whereas FimE-catalyzed switching is on to off only. We show here that under liquid shaking growth conditions, Nissle 1917 did not express type 1 fimbriae, due to a truncation of the
fimB
gene by an 1,885-bp insertion element. Despite its
fimB
null status, Nissle 1917 was still capable of off-to-on switching of the phase switch and expressing type 1 fimbriae when grown under static conditions. This phase switching was not catalyzed by FimE, by truncated FimB, or by information residing within the insertion element. No further copies of
fimB
seemed to be present on the chromosome of Nissle 1917, suggesting that another tyrosine recombinase in Nissle 1917 is responsible for the low-frequency off-to-on inversion of the phase switch that is strongly favored under static growth conditions. This is the first report documenting the non-FimB- or non-FimE-catalyzed inversion of the
fim
switch.
Multiple Control of Flagellum Biosynthesis in
Escherichia coli
: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the
flhDC
Master Operon
Authors: Soutourina O. , Kolb A. , Krin E. , Laurent-Winter C. , Rimsky S. , Danchin A. , Bertin P.
Published: December 2019

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that
crp
and
hns
mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of
fliA
and
fliC
, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the
flhDC
master operon restored, at least in part, motility in
crp
and
hns
mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates
flhDC
expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the
flhDC
promoter was decreased in the
crp
strain and increased in the
hns
mutant. In contrast, the activity of a transcriptional fusion encompassing the entire
flhDC
regulatory region extending to the ATG translational start codon was strongly reduced in both
hns
and
crp
mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a
crp
mutant by activation-deficient CAP mutated proteins and characterization of
cfs
, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates
flhDC
transcription by binding to its promoter and interacting with RNA polymerase.
CDC64
Encodes Cytoplasmic Alanyl-tRNA Synthetase, Ala1p, of
Saccharomyces cerevisiae
Authors: Wrobel Carolyn , Schmidt Emmett V. , Polymenis Michael
Published: December 2019

The
cdc64-1
mutation causes G
1
arrest in
Saccharomyces cerevisiae
corresponding to a type II Start phenotype. We report that
CDC64
encodes Ala1p, an alanyl-tRNA synthetase. Thus,
cdc64-1
might affect charging of tRNA
Ala
and thereby initiation of cell division.
Structural and Functional Analyses of the Secondary Cell Wall Polymer of
Bacillus sphaericus
CCM 2177 That Serves as an S-Layer-Specific Anchor
Authors: Ilk Nicola , Kosma Paul , Puchberger Michael , Egelseer Eva M. , Mayer Harald F. , Sleytr Uwe B. , Sára Margit
Published: December 2019

Sacculi of
Bacillus sphaericus
CCM 2177 contain a secondary cell wall polymer which was completely extracted with 48% hydrofluoric acid. Nuclear magnetic resonance analysis showed that the polymer is composed of repeating units, as follows: →3)-[4,6-
O
-(1-carboxyethylidene)]
∼0.5
-β-
d
-Man
p
NAc-(1→4)-β-
d
-Glc
p
NAc-(1→. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.
Characterization of IS
1547
, a New Member of the IS
900
Family in the
Mycobacterium tuberculosis
Complex, and Its Association with IS
6110
Authors: Fang Z. , Doig C. , Morrison N. , Watt B. , Forbes K. J.
Published: December 2019

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS
116
, IS
900
, IS
901
, and IS
1110
have been proposed as members of the IS
900
family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS
1547
, which was first identified in a clinical isolate of
Mycobacterium tuberculosis
. Its structure, insertion site, and putative transposase all conform with the conventions of the IS
900
family, suggesting that it is a new member of this family. IS
1547
was detected only in isolates of the
M. tuberculosis
complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the
M. tuberculosis
complex and for distinguishing different strains of
M. tuberculosis
.
ipl
is a preferential locus for IS
6110
insertion where there are eight known different insertion sites for IS
6110
. Surprisingly, the DNA sequence of
ipl
is now known to be a part of IS
1547
, meaning that IS
1547
is a preferential site for IS
6110
insertion.
Sporadic Distribution of tRNA
CCU
Arg
Introns among α-Purple Bacteria: Evidence for Horizontal Transmission and Transposition of a Group I Intron
Authors: Paquin Bruno , Heinfling Annette , Shub David A.
Published: December 2019

A group I intron interrupts the tRNA
CCU
Arg
gene of the α-purple bacterium
Agrobacterium tumefaciens
(B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173–176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of α-purple bacteria. Of 10 newly identified tRNA
CCU
Arg
genes, we found only two that contained an intron homologous to that of the
Agrobacterium
tRNA
CCU
Arg
intron. This restricted and scattered distribution of the tRNA
CCU
Arg
intron among α-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA
UAA
Leu
introns found in strains of the cyanobacterium
Microcystis aeruginosa
(K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293–298, 1997) and α-purple tRNA
CCU
Arg
introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA
UAA
Leu
introns.
The
merG
Gene Product Is Involved in Phenylmercury Resistance in
Pseudomonas
Strain K-62
Authors: Kiyono Masako , Pan-Hou Hidemitsu
Published: December 2019

The physiological function of a new gene, hereby designated
merG
, located between
merA
and
merB
on the broad-spectrum
mer
operon of
Pseudomonas
strain K-62 plasmid pMR26 was investigated. The 654-bp
merG
gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the
merG
gene from the broad-spectrum
mer
operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain.
Escherichia coli
cells bearing pMU29, which carries a deletion of the
merG
gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the
merG
gene in a compatible plasmid was transformed into the
E. coli
strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the
merG
gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.
Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of
Nocardioides
sp. Strain KP7
Authors: Adachi Kyoko , Iwabuchi Tokuro , Sano Hiroshi , Harayama Shigeaki
Published: December 2019

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in
Nocardioides
sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (
E
)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name,
trans
-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by
1
H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.
Functional Analysis of a Rickettsial OmpA Homology Domain of
Shigella flexneri
IcsA
Authors: Charles Macarthur , Magdalena Juana , Theriot Julie A. , Goldberg Marcia B.
Published: December 2019

Shigella flexneri
is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa
S. flexneri
outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of
S. flexneri
and
Escherichia coli
that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione
S
-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.
Cra-Dependent Transcriptional Activation of the
icd
Gene of
Escherichia coli
Authors: Prost Jean-François , Nègre Didier , Oudot Christelle , Murakami Katsuhiko , Ishihama Akira , Cozzone Alain J. , Cortay Jean-Claude
Published: December 2019

The
icd
gene of
Escherichia coli
, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified
icd
P1 and a newly detected second promoter,
icd
P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the
icd
control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position −76.5 within the
icd
P2 promoter region and (ii) three particular mutations in the C-terminal end of the α subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the
icd
P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.
Effect of
wzx
(
rfbX
) Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis in
Pseudomonas aeruginosa
O5
Authors: Burrows Lori L. , Lam Joseph S.
Published: December 2019

The
wbp
cluster of
Pseudomonas aeruginosa
O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly
wbpF
), contiguous with
wzy
in the
wbp
cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or “flippase.” Insertion of a Gm
r
cassette at two separate sites within the putative
wzx
gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal
wzx
gene replacement mutations. Surprisingly, inactivation of
wzx
also led to a marked delay in production of the ATP-binding cassette–transporter-dependent,
d
-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in
trans
. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in
P. aeruginosa
O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103–1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of
wzx
mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.
Cell Density-Dependent Starvation Survival of
Rhizobium leguminosarum
bv. phaseoli: Identification of the Role of an N- Acyl Homoserine Lactone in Adaptation to Stationary-Phase Survival
Authors: Thorne Stephen H. , Williams Huw D.
Published: December 2019

The cell density dependence of stationary-phase survival of
Rhizobium leguminosarum
has been investigated. Following starvation by exhaustion of carbon or nitrogen, but not of phosphorus, the survival of cultures was dependent on the cell density at entry into stationary phase. High-density cultures survived with little or no loss of viability over a 20-day period in stationary phase. In contrast, low-density cultures lost viability rapidly but consisted of a heterogeneous population, a small fraction of which successfully adapted and eventually formed a stable, surviving population. The threshold density above which the cultures survived successfully in stationary phase was dependent on the growth conditions and the strain used. We took advantage of the fact that
R. leguminosarum
survives poorly following starvation by resuspension in carbon-free medium to demonstrate that cell density-dependent survival was mediated by a component accumulating in the growth medium. The effects of this medium component on survival in resuspension assays could be mimicked by an
N
-acyl homoserine lactone,
N
-(3
R
-hydroxy-7-
cis
-tetradecanoyl)-
l
-homoserine lactone, previously demonstrated to have a role in controlling cell density-dependent phenomena in
R. leguminosarum
. The Sym plasmids pRP2JI and pRL1JI were found to be essential for the production of the extracellular factor, which could also be made in
Escherichia coli
carrying the cosmid clone pIJ1086 containing a specific region of pRL1JI.
The Signal Transduction Protein GlnK Is Required for NifL-Dependent Nitrogen Control of
nif
Gene Expression in
Klebsiella pneumoniae
Authors: Jack Rachael , De Zamaroczy Miklos , Merrick Mike
Published: December 2019

In
Klebsiella pneumoniae
, transcription of the nitrogen fixation (
nif
) genes is regulated in response to molecular oxygen or availability of fixed nitrogen by the coordinated activities of the
nifA
and
nifL
gene products. NifA is a
nif
-specific transcriptional activator, the activity of which is inhibited by interaction with NifL. Nitrogen control of NifL occurs at two levels: transcription of the
nifLA
operon is regulated by the global
ntr
system, and the inhibitory activity of NifL is controlled in response to fixed nitrogen by an unknown factor.
K. pneumoniae
synthesizes two P
II
-like signal transduction proteins, GlnB, which we have previously shown not to be involved in the response of NifL to fixed nitrogen, and the recently identified protein GlnK. We have now cloned the
K. pneumoniae glnK
gene, studied its expression, and shown that a null mutation in
glnK
prevents NifL from responding to the absence of fixed nitrogen, i.e., from relieving the inhibition of NifA activity. Hence, GlnK appears to be involved, directly or indirectly, in NifL-dependent regulation of
nif
gene expression in
K. pneumoniae
. Comparison of the GlnB and GlnK amino acid sequences from six species of proteobacteria identifies five residues (residues 3, 5, 52, 54, and 64) which serve to distinguish the GlnB and GlnK proteins.
An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ma Kesen , Adams Michael W. W.
Published: December 2019

Pyrococcus furiosus
is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO
2
, and H
2
, together with minor amounts of ethanol. The organism also generates H
2
S in the presence of elemental sulfur (S
0
). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S
0
. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent

K
m

s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively.
P. furiosus
alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.
Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
: the Third of a Putative Five-Member Tungstoenzyme Family
Authors: Roy Roopali , Mukund Swarnalatha , Schut Gerrit J. , Dunn Dianne M. , Weiss Robert , Adams Michael W. W.
Published: December 2019

Pyrococcus furiosus
is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO
2
, and H
2
. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from
P. furiosus
. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS
−
) under reducing conditions. FOR uses
P. furiosus
ferredoxin as an electron acceptor (

K
m

= 100 μM) and oxidizes a range of aldehydes. Formaldehyde (

K
m

= 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C
5
semi- or dialdehyde, e.g., glutaric dialdehyde (

K
m

= 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (
for
) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the
P. furiosus
chromosome. Two additional (and nonlinked) genes (termed
wor4
and
wor5
) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.
Active Efflux and Diffusion Are Involved in Transport of
Pseudomonas aeruginosa
Cell-to-Cell Signals
Authors: Pearson James P. , Van Delden Christian , Iglewski Barbara H.
Published: December 2019

Many gram-negative bacteria communicate by
N
-acyl homoserine lactone signals called autoinducers (AIs). In
Pseudomonas aeruginosa
, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (ς
s
), and biofilm differentiation. The fact that a similar signal,
N
-(3-oxohexanoyl) homoserine lactone, freely diffuses through
Vibrio fischeri
and
Escherichia coli
cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two
P. aeruginosa
AIs,
N
-(3-oxododecanoyl) homoserine lactone (3OC
12
-HSL) (formerly called PAI-1) and
N
-butyryl homoserine lactone (C
4
-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [
3
H]C
4
-HSL was added to cell suspensions of
P. aeruginosa
, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [
3
H]3OC
12
-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [
3
H]3OC
12
-HSL, suggesting the involvement of active efflux. A defined mutant lacking the
mexA-mexB-oprM
-encoded active-efflux pump accumulated [
3
H]3OC
12
-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C
4
-HSL,
P. aeruginosa
cells are not freely permeable to 3OC
12
-HSL. Instead, the
mexA-mexB-oprM
-encoded efflux pump is involved in active efflux of 3OC
12
-HSL. Apparently the length and/or degree of substitution of the
N
-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by
P. aeruginosa
.
Inhibition of Homologous Recombination by the Plasmid MucA′B Complex
Authors: Venderbure Claude , Chastanet Arnaud , Boudsocq François , Sommer Suzanne , Bailone Adriana
Published: December 2019

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F
−
recipients expressing MucA′B from a P

tac

promoter, Hfr × F
−
recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F
lac
plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in
trans
-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per
lexA
(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.
Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of
Pseudomonas putida
GJ31
Authors: Mars Astrid E. , Kingma Jaap , Kaschabek Stefan R. , Reineke Walter , Janssen Dick B.
Published: December 2019

Pseudomonas putida
GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (
cbzE
) was cloned and sequenced. The
cbzE
gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a
meta
-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930–5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2,3-dioxygenase of
P. putida
UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.
The Lethal Effect of a Benzamide Derivative, 3-Methoxybenzamide, Can Be Suppressed by Mutations within a Cell Division Gene,
ftsZ
, in
Bacillus subtilis
Authors: Ohashi Yoshiaki , Chijiiwa Yoshie , Suzuki Koichiro , Takahashi Kouki , Nanamiya Hideaki , Sato Tsutomu , Hosoya Yoshiko , Ochi Kozo , Kawamura Fujio
Published: December 2019

3-Methoxybenzamide (3-MBA), which is known to be an inhibitor of ADP-ribosyltransferase, inhibits cell division in
Bacillus subtilis
, leading to filamentation and eventually lysis of cells. Our genetic analysis of 3-MBA-resistant mutants indicated that the primary target of the drug is the cell division system involving FtsZ function during both vegetative growth and sporulation.
Components of the
Salmonella
Flagellar Export Apparatus and Classification of Export Substrates
Authors: Minamino Tohru , Macnab Robert M.
Published: December 2019

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive
fliQ
or
fliR
mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.
An Lrp-Like Protein of the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Which Binds to Its Own Promoter
Authors: Napoli Alessandra , van der Oost John , Sensen Christoph W. , Charlebois Robert L. , Rossi Mosé , Ciaramella Maria
Published: December 2019

Regulation of gene expression in the domain
Archaea
, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain
Archaea
(i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in
Archaea
. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon
Sulfolobus solfataricus
. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class
Bacteria
. We show that Lrs14, expressed in
Escherichia coli
, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the
E. coli
Lrp, Lrs14 is autoregulated. We also show that the
lrs14
transcript is accumulated in the late growth stages of
S. solfataricus
.
SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions
Authors: Rahman M. Sayeedur , Humayun M. Zafri
Published: December 2019

Escherichia coli
cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N
4
-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective
E. coli
strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous
lac
-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.