bluuebunny/crossref_metadata_2025 huggingface dataset:
Thickness and Elasticity of Gram-Negative Murein Sacculi Measured by Atomic Force Microscopy
Authors: Yao X. , Jericho M. , Pink D. , Beveridge T.
Published: December 2019

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi fromEscherichia coliK-12 andPseudomonas aeruginosaPAO1. Air-dried sacculi fromE. colihad a thickness of 3.0 nm, whereas those fromP. aeruginosawere 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. WhenE. colisacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 × 107N/m2was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 × 108to 4 × 108N/m2and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intactE. colisacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927–953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301–366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.
Catabolite Regulation of the
pta
Gene as Part of Carbon Flow Pathways in
Bacillus subtilis
Authors: Presecan-Siedel Elena , Galinier Anne , Longin Robert , Deutscher Josef , Danchin Antoine , Glaser Philippe , Martin-Verstraete Isabelle
Published: December 2019

In
Bacillus subtilis
, the products of the
pta
and
ackA
genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated
ackA
did not grow in the presence of glucose. Inactivation of
pta
had only a weak inhibitory effect on growth. In contrast to
pta
and
ackA
in
Escherichia coli
, the corresponding
B. subtilis
genes are not cotranscribed. Expression of the
pta
gene was increased in the presence of glucose, as has been reported for
ackA
. The effects of the predicted
cis
-acting catabolite response element (CRE) located upstream from the promoter and of the
trans
-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of
pta
were investigated. As for
ackA
, glucose activation was abolished in
ccpA
and
hprK
mutants and in the
ptsH1 crh
double mutant. Footprinting experiments demonstrated an interaction between CcpA and the
pta
CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the
pta
gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.
Characterization of a Periplasmic ATP-Binding Cassette Iron Import System of
Brachyspira
(
Serpulina
)
hyodysenteriae
Authors: Dugourd Dominique , Martin Christine , Rioux Clément R. , Jacques Mario , Harel Josée
Published: December 2019

The nucleotide sequence of the pathogenic spirochete
Brachyspira hyodysenteriae bit
(for “
Brachyspira
iron transport”) genomic region has been determined. The
bit
region is likely to encode an iron ATP-binding cassette transport system with some homology to those encountered in gram-negative bacteria. Six open reading frames oriented in the same direction and physically linked have been identified. This system possesses a protein containing ATP-binding motifs (BitD), two hydrophobic cytoplasmic membrane permeases (BitE and BitF), and at least three lipoproteins (BitA, BitB, and BitC) with homology to iron periplasmic binding proteins. These periplasmic binding proteins exhibit lipoprotein features. They are labeled by [
3
H]palmitate when tested in recombinant
Escherichia coli
, and their signal peptides are typical for substrates of the type II secretory peptidase. The FURTA system and Congo red assay indicate that BitB and BitC are involved in iron binding. The Bit system is detected only in
B. hyodysenteriae
and is absent from
B. innocens
and
B. pilosicoli
.
The Respiratory System and Diazotrophic Activity of
Acetobacter diazotrophicus
PAL5
Authors: Flores-Encarnación M. , Contreras-Zentella M. , Soto-Urzua L. , Aguilar G. R. , Baca B. E. , Escamilla J. E.
Published: December 2019

The characteristics of the respiratory system of
Acetobacter diazotrophicus
PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min
−1
liter of medium
−1
) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome
c
oxidase-type activity was poor as measured by reduced
N
,
N
,
N
,
N
′-tetramethyl-
p
-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome
ba
as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in
c
-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized
c
-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH
4
)
2
SO
4
caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase
ba
decreased 6.8- and 10-fold, respectively. In these cells, a
bd
-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome
ba
are key components of the respiratory system of
A. diazotrophicus
during aerobic diazotrophy.
Control of Synthesis and Secretion of the
Bacillus subtilis
Protein YqxM
Authors: Stöver Axel G. , Driks Adam
Published: December 2019

yqxM
is a
Bacillus subtilis
gene of unknown function residing in an operon with
sipW
, which encodes a signal peptidase, and
tasA
, which encodes an antibiotic protein secreted in a
sipW
-dependent manner. YqxM was undetectable during growth in a variety of rich media, including Luria-Bertani (LB) medium, or in minimal media or under heat shock or ethanol stress conditions but was synthesized and secreted during growth in LB medium supplemented with 1.2 M NaCl. Consistent with the possible involvement of
sipW
in YqxM secretion, inactivation of
sipW
prevented YqxM secretion. YqxM was produced and secreted in a
sipW
-dependent manner during growth in LB medium when the sequences upstream of
yqxM
were replaced with those of the inducible P

spac

promoter. Coexpression of
yqxM
and
sipW
in
Escherichia coli
resulted in a decrease in the apparent molecular mass of YqxM, consistent with the removal of a signal peptide. These experiments suggest that YqxM production is induced by a high concentration of salt and that YqxM is secreted under the control of SipW. We hypothesize that during most conditions of growth, YqxM is present at very low levels or is not synthesized at all and that this low level or absence is due, at least in part, to posttranscriptional repression.
PHR1
and
PHR2
of
Candida albicans
Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans
Authors: Fonzi William A.
Published: December 2019

PHR1
and
PHR2
encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen
Candida albicans
. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and β-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between β-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in β-1,3-glucan-associated β-1,6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between β-1,6- and β-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process β-1,3-glucans and make available acceptor sites for the attachment of β-1,6-glucans.
Mercury Resistance in
Bacillus cereus
RC607: Transcriptional Organization and Two New Open Reading Frames
Authors: Gupta Amit , Phung Le T. , Chakravarty Leena , Silver Simon
Published: December 2019

The chromosomal mercury resistance determinant of
Bacillus cereus
RC607 confers resistance to inorganic mercury and to organomercurials. The order of genes in the completed mercury resistance determinant is operator-promoter 1 (O/P1)
merR1 merT
open reading frame 3 (ORF3) ORF4
merA
O/P2
merR2 merB2 merB1
. The previously undetermined 1-kb DNA sequence between the
merA
and
merB1
genes includes two significant ORFs, whose predicted protein products are homologous with MerR (the transcriptional regulator) and MerB (the organomercurial lyase enzyme). Two transcriptional start sites (promoters), O/P1 at the beginning of the determinant and O/P2 immediately upstream of the sixth ORF, the newly identified
merR2
, were mapped by reverse transcriptase (RT) primer extension. A long 6.3-kb mRNA traversing all eight ORFs was shown by RT-PCR. Growth sensitivity measurements in liquid media and cellular mercury volatization assays characterized inducibility and differences in functional activity in
B. cereus
RC607 and after cloning of the
mer
determinant into plasmids in
Escherichia coli
.
Transcriptional Analysis of the Rubrerythrin and Superoxide Dismutase Genes of
Clostridium perfringens
Authors: Geissmann Thomas A. , Teuber Michael , Meile Leo
Published: December 2019

We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from
Clostridium perfringens
containing the superoxide dismutase-encoding gene,
sod
. Previously, rubrerythrin from
C. perfringens
had been isolated and its gene (
rbr
) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152–7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of
rbr
and
sod
of
C. perfringens
. Thus,
rbr
and
sod
each represent a monocistronic operon. Their transcription start points were located by primer extension analyses.
sod
transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither
sod
nor
rbr
transcription was influenced by oxidative stress.
Bacillus subtilis yckG
and
yckF
Encode Two Key Enzymes of the Ribulose Monophosphate Pathway Used by Methylotrophs, and
yckH
Is Required for Their Expression
Authors: Yasueda Hisashi , Kawahara Yoshio , Sugimoto Shin-ichi
Published: December 2019

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumed
yckG
gene product (YckG) of
Bacillus subtilis
shows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249–256, 1997). We have also investigated the sequence similarity between the
yckF
gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125–130, 1999) and found that the
yckG
and
yckF
genes of
B. subtilis
express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced in
B. subtilis
by formaldehyde, with induction showing dependence on the
yckH
gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in
B. subtilis
. In conclusion, we found an active
yckG
(for HPS)-
yckF
(for PHI) gene structure (now named
hxlA-hxlB
) in a nonmethylotroph,
B. subtilis
, which inherently preserves the RuMP pathway.
Renaturation of RecombinantTreponema pallidumRare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation
Authors: Zhang Hongwei H. , Blanco David R. , Exner Maurice M. , Shang Ellen S. , Champion Cheryl I. , Phillips Martin L. , Miller James N. , Lovett Michael A.
Published: December 2019

We have previously observed that while nativeTreponema pallidumrare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 Å cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed inEscherichia coliand purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylasebprotein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 Å cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.
Interference with Murein Turnover Has No Effect on Growth but Reduces β-Lactamase Induction in
Escherichia coli
Authors: Kraft Angelika R. , Prabhu Julia , Ursinus Astrid , Höltje Joachim-Volker
Published: December 2019

Physiological studies of a mutant of
Escherichia coli
lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC β-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of β-lactamase, present on a plasmid carrying the
ampC
and
ampR
genes from
Enterobacter cloacae
. Overproduction of MltB stimulated β-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed
ampC
expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like β-lactamases.
Genetic Dissection of the Outer Membrane Secretin PulD: Are There Distinct Domains for Multimerization and Secretion Specificity?
Authors: Guilvout Ingrid , Hardie Kim R. , Sauvonnet Nathalie , Pugsley Anthony P.
Published: December 2019

Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of
Klebsiella oxytoca
. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic β strands in the C-terminal half of the polypeptide (the β domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the β domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in
trans
. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the β domain was present in
trans
. We conclude that the β domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of
Erwinia chrysanthemi
without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by
E. chrysanthemi
(V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007–3016, 1997).
Identification of the
miaB
Gene, Involved in Methylthiolation of Isopentenylated A37 Derivatives in the tRNA of
Salmonella typhimurium
and
Escherichia coli
Authors: Esberg Birgitta , Leung Hon-Chiu Eastwood , Tsui Ho-Ching Tiffany , Björk Glenn R. , Winkler Malcolm E.
Published: December 2019

The tRNA of the
miaB2508
::Tn
10d
Cm mutant of
Salmonella typhimurium
is deficient in the methylthio group of the modified nucleoside
N
6
-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms
2
io
6
A37). By sequencing, we found that the Tn
10d
Cm of this strain had been inserted into the
f474
(
yleA
) open reading frame, which is located close to the
nag
locus in both
S. typhimurium
and
Escherichia coli
. By complementation of the
miaB2508
::Tn
10d
Cm mutation with a minimal subcloned
f474
fragment, we showed that
f474
could be identified as the
miaB
gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of
miaB
in
E. coli
and
S. typhimurium
. A Rho-independent terminator was identified downstream of the
miaB
gene, at which the majority (96%) of the
miaB
transcripts terminate in
E. coli
, showing that the
miaB
gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms
2
i(o)
6
A37 formation.
RNA Polymerase α and ς
70
Subunits Participate in Transcription of the
Escherichia coli uhpT
Promoter
Authors: Olekhnovich Igor N. , Kadner Robert J.
Published: December 2019

Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of the
Escherichia coli uhpT
gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the
uhpT
promoter between positions −80 and −32 upstream of the transcription start site, and CAP binds to a single site centered at position −103.5. The role in
uhpT
transcription of portions of RNA polymerase Eς
70
holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of a
uhpT-lacZ
transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the −35 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient
uhpT
expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (αCTD) were important for
uhpT
transcription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional αCTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.
General Nitrogen Regulation of Nitrate Assimilation Regulatory Gene
nasR
Expression in
Klebsiella oxytoca
M5al
Authors: Wu Stephen Qitu , Chai Weihang , Lin Janine T. , Stewart Valley
Published: December 2019

Klebsiella oxytoca
can assimilate nitrate and nitrite by using enzymes encoded by the
nasFEDCBA
operon. Expression of the
nasF
operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of
nasR
gene expression. We constructed strains bearing single-copy Φ(
nasR-lacZ
) operon fusions within the chromosomal
rhaBAD-rhaSR
locus. The expression of Δ
rhaBS
::[Φ(
nasR-lacZ
)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both
ntrC
and
rpoN
null mutants, indicating that Ntr control of
nasR
gene expression requires the NtrC and ς
N
(ς
54
) proteins. Sequence inspection of the
nasR
control region reveals an apparent ς
N
-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the ς
N
-dependent
nasR
promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of β-galactosidase expression indistinguishable from Φ(
nasR-lacZ
) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of
nasR
transcription. Together, these results indicate that
nasR
gene expression does not require specific upstream NtrC-binding sequences, as previously noted for
argT
gene expression in
Salmonella typhimurium
(G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107–117, 1988).
Negative Control of Flagellum Synthesis in
Pseudomonas aeruginosa
Is Modulated by the Alternative Sigma Factor AlgT (AlgU)
Authors: Garrett Edward S. , Perlegas Demetra , Wozniak Daniel J.
Published: December 2019

Many respiratory isolates of
Pseudomonas aeruginosa
from cystic fibrosis patients are mucoid (alginate producing) yet lack flagella. It was hypothesized that an alginate regulator inhibits flagellar gene expression. Mutations in
algB
,
algR
, and
algT
resulted in nonmucoid derivatives, yet
algT
mutants expressed flagella. AlgT-dependent control of flagellum synthesis occurred through inhibition of
fliC
but not
rpoN
transcription.
Characterization of a Novel, Antifungal, Chitin-Binding Protein from
Streptomyces tendae
Tü901 That Interferes with Growth Polarity
Authors: Bormann Christiane , Baier Daniel , Hörr Ingmar , Raps Claudia , Berger Jürgen , Jung Günther , Schwarz Heinz
Published: December 2019

The
afp1
gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing
Streptomyces tendae
Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against
Paecilomyces variotii
in agar diffusion assays. The 384-bp
afp1
gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for
Streptomyces
genes. AFP1 was purified from culture filtrate of
S. tendae
carrying the
afp1
gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of
P. variotii
but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of
Aspergillus
species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.
Molecular Analysis of the
Candida albicans
Homolog of
Saccharomyces cerevisiae MNN9
, Required for Glycosylation of Cell Wall Mannoproteins
Authors: Southard Susan B. , Specht Charles A. , Mishra Chitra , Chen-Weiner Joan , Robbins Phillips W.
Published: December 2019

The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the
Candida albicans
homolog of
Saccharomyces cerevisiae MNN9
, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption.
CaMNN9
has significant homology with
S. cerevisiae MNN9
, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in
Candida. CaMNN9
resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of
CaMNN9
leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with β-1,3-glucanase. Like all members of the
S. cerevisiae MNN9
gene family the
Camnn9
Δ strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the
Camnn9
Δ strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of
MNN9
gene family members
CaVAN1
and
CaANP1
in the
Camnn9
Δ strain showed that transcription of those genes is not affected in the absence of
CaMNN9
transcription. Our results suggest that, while the role
MNN9
plays in glycosylation in both
Candida
and
Saccharomyces
is conserved, loss of
MNN9
function in
C. albicans
leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target.
Citrate Synthase Mutants of
Sinorhizobium meliloti
Are Ineffective and Have Altered Cell Surface Polysaccharides
Authors: Mortimer Michael W. , McDermott Timothy R. , York Gregory M. , Walker Graham C. , Kahn Michael L.
Published: December 2019

The
gltA
gene, encoding
Sinorhizobium meliloti
104A14 citrate synthase, was isolated by complementing an
Escherichia coli gltA
mutant. The
S. meliloti gltA
gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type
gltA
with the
gltA
::
kan
allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0.2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.
Mutations in Catabolite Control Protein CcpA Showing Glucose-Independent Regulation in
Bacillus megaterium
Authors: Küster-Schöck Elke , Wagner Andrea , Völker Uwe , Hillen Wolfgang
Published: December 2019

We identified five single amino acid exchanges in CcpA that lead to permanent repression of the xylose utilization genes in the absence of glucose. Other proteins from the CcpA regulon also show glucose-independent regulation in the mutants. The mutant CcpA proteins bind to the DNA target catabolite responsive elements without the corepressor HPr-Ser-P.
The Periplasmic 9.6-Kilodaltonc-Type Cytochrome ofGeobacter sulfurreducensIs Not an Electron Shuttle to Fe(III)
Authors: Lloyd Jon R. , Blunt-Harris Elizabeth L. , Lovley Derek R.
Published: December 2019

Geobacter sulfurreducenscontains a 9.6-kDac-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions ofG. sulfurreducensit did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellularc-type cytochromes in cultures ofG. sulfurreducensdemonstrated that the dominantc-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.
Topology of RbsC, a Membrane Component of the Ribose Transporter, Belonging to the AraH Superfamily
Authors: Park Yongkyu , Park Chankyu
Published: December 2019

RbsC of
Escherichia coli
is the hydrophobic membrane component of ribose uptake system classified as the ATP-binding cassette transporter. To understand the structure and function of RbsC, its transmembrane topology was investigated by using 64 RbsC-PhoA fusions isolated either specifically or randomly. In order to confirm the cytoplasmic location of the short C-terminal region (5 amino acids), inside-out or right-side-out membrane vesicles were generated, and the C-terminal region was found to be digested by carboxypeptidase A only in inside-out vesicles. This result is consistent with the model, based on the results of alkaline phosphatase fusions, in which the protein traverses the membrane six times and the N and C termini are exposed to the cytoplasm.
Escherichia coli
Cells Exposed to Streptomycin Display a Mutator Phenotype
Authors: Ren Li , Rahman M. Sayeedur , Humayun M. Zafri
Published: December 2019

Mistranslation mediated by the
mutA
and
mutC
tRNA alleles elicits a strong mutator phenotype (H. S. Murphy and M. Z. Humayun, J. Bacteriol. 179:7507–7514, 1997; M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380–4385, 1996). Here, we show that exposure to streptomycin, an antibiotic known to promote mistranslation, induces a
recA
- and
umuDC
-independent mutator phenotype detected as enhanced mutagenesis at a 3,N
4
-ethenocytosine lesion borne on transfected M13 single-stranded DNA.
The Presence of Glutamate Dehydrogenase Is a Selective Advantage for the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 under Nonexponential Growth Conditions
Authors: Chávez S. , Lucena J. M. , Reyes J. C. , Florencio F. J. , Candau P.
Published: December 2019

The unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to
Synechocystis
sp. strain PCC 6803 in late stages of growth.
The
mutK
Gene of
Vibrio cholerae
: a New Gene Involved in DNA Mismatch Repair
Authors: Bhakat Kishor K. , M. Sudarshana Sharma , Das Jyotirmoy
Published: December 2019

A new gene,
mutK
, of
Vibrio cholerae
, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the
mutK
gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of
Escherichia coli mutS
,
mutL
,
mutU
, and
dam
mutants. The spontaneous mutation frequency of a chromosomal
mutK
knockout mutant was almost identical to that of wild-type
V. cholerae
cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the
mutK
gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the
mutK
gene product. The
mutK
gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of
mutK
, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the
E. coli vsr
gene. ORF1, located farther upstream of
mutK
, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of
mutK
, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The
mutK
gene has recently been positioned in the ordered cloned DNA map of the genome of the
V. cholerae
strain from which the gene was isolated (10).
Recovery of DNA Replication in UV-Irradiated
Escherichia coli
Requires both Excision Repair and RecF Protein Function
Authors: Courcelle Justin , Crowley David J. , Hanawalt Philip C.
Published: December 2019

After UV doses that disrupt DNA replication, the recovery of replication at replication forks in
Escherichia coli
requires a functional copy of the
recF
gene. In
recF
mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that
recF
function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of
recF
to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair,
recF
+
cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either
uvr
or
recF
result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that
recF
and excision repair contribute to a common pathway in the recovery of replication.
Phenotypic Analysis of Random
hns
Mutations Differentiate DNA-Binding Activity from Properties of
fimA
Promoter Inversion Modulation and Bacterial Motility
Authors: Donato Gina M. , Kawula Thomas H.
Published: December 2019

H-NS is a major
Escherichia coli
nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in
hns
result in constitutive expression of the
proU
and
fimB
genes, increased
fimA
promoter inversion rates, and repression of the
flhCD
master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned
hns
gene into a mutator strain and collected a series of mutant alleles that failed to repress
proU
expression. Each of these isolated
hns
mutant alleles also failed to repress
fimB
expression, suggesting that H-NS-specific repression of
proU
and
fimB
occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type
fimA
promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various
hns
mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.
High-Molecular-Mass Multi-
c
-Heme Cytochromes from
Methylococcus capsulatus
Bath
Authors: Bergmann David J. , Zahn James A. , DiSpirito Alan A.
Published: December 2019

The polypeptide and structural gene for a high-molecular-mass
c
-type cytochrome, cytochrome
c
553O
, was isolated from the methanotroph
Methylococcus capsulatus
Bath. Cytochrome
c
553O
is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme
c
concentration was estimated to be 8.2 ± 0.4 mol of heme
c
per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome
c
553O
was used to identify a DNA fragment from
M. capsulatus
Bath that contains
occ
, the gene encoding cytochrome
c
553O
.
occ
is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic
c
-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with
occ
and contains nine
c
-heme-binding motifs. ORF3 encodes a putative periplasmic
c
-type cytochrome with a molecular mass of 94,000 Da and contains seven
c
-heme-binding motifs but shows no sequence homology to
occ
or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome
c
553O
and the gene products of ORF1 and ORF3 constitute a new class of
c
-type cytochrome.
Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic
Crenarchaeota
and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation
Authors: Menendez Castor , Bauer Zsuzsa , Huber Harald , Gad’on Nasser , Stetter Karl-Otto , Fuchs Georg
Published: December 2019

The pathway of autotrophic CO
2
fixation was studied in the phototrophic bacterium
Chloroflexus aurantiacus
and in the aerobic thermoacidophilic archaeon
Metallosphaera sedula
. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO
2
-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either
14
CO
2
or [
14
C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for
C. aurantiacus
. The investigation was extended to the autotrophic archaea
Sulfolobus metallicus
and
Acidianus infernus
, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as
C. aurantiacus
, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea
M. sedula
,
S. metallicus
, and
A. infernus
use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO
2
fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in
C. aurantiacus
.