bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of the Nitrite-Reducing System of
Staphylococcus carnosus
Authors: Neubauer H. , Pantel I. , Götz F.
Published: December 2019

Characterization of a nitrite reductase-negative
Staphylococcus carnosus
Tn
917
mutant led to the identification of the
nir
operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for
nir
promoter activity. In the absence of NirR, a weak promoter upstream of
sirA
seems to drive transcription of
sirA
,
nirB
,
nirD
, and
sirB
in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the
nir
promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the
nir
operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene,
nirC
, is located upstream of the
nir
operon.
nirC
encodes a putative integral membrane-spanning protein of unknown function. A
nirC
mutant showed no distinct phenotype.
Environmental Signals Modulate ToxT-Dependent Virulence Factor Expression in
Vibrio cholerae
Authors: Schuhmacher Darren A. , Klose Karl E.
Published: December 2019

The regulatory protein ToxT directly activates the transcription of virulence factors in
Vibrio cholerae
, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of
toxT
. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in
V. cholerae
at 30°C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37°C. Also, expression of six ToxT-dependent Tn
phoA
fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by
ctxAp
- and
tcpAp
-luciferase fusions confirmed that negative regulation by 37°C or bile occurs at the transcriptional level in
V. cholerae
. Interestingly, ToxT-dependent transcription of these same promoters in
Salmonella typhimurium
was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in
V. cholerae
and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.
Protein ProQ Influences Osmotic Activation of Compatible Solute Transporter ProP in
Escherichia coli
K-12
Authors: Kunte H. Jörg , Crane Rebecca A. , Culham Doreen E. , Richmond Deborah , Wood Janet M.
Published: December 2019

ProP is an osmoregulatory compatible solute transporter in
Escherichia coli
K-12. Mutation
proQ220
::Tn
5
decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter
proP
transcription or the ProP protein level. Allele
proQ220
::Tn
5
was isolated, and the
proQ
sequence was determined. Locus
proQ
is upstream from
prc
(
tsp
) at 41.2 centisomes on the genetic map. The
proQ220
::Tn
5
and
prc
phenotypes were different, however. Gene
proQ
is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified
proQ
into vector pBAD24 produced a plasmid containing the wild-type
proQ
open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in
proQ
+
bacteria but not in
proQ220
::Tn
5
bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.
Correlation of Activity Regulation and Substrate Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase Activity in
Rhodospirillum rubrum
Authors: Kim Kitai , Zhang Yaoping , Roberts Gary P.
Published: December 2019

In
Rhodospirillum rubrum
, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.
Role of the Alternative Sigma Factor ς
S
in Expression of the AlkS Regulator of the
Pseudomonas oleovorans
Alkane Degradation Pathway
Authors: Canosa Inés , Yuste Luis , Rojo Fernando
Published: December 2019

The AlkS protein activates transcription from the
PalkB
promoter, allowing the expression of a number of genes required for the assimilation of alkanes in
Pseudomonas oleovorans
. We have identified the promoter from which the
alkS
gene is transcribed,
PalkS
, and analyzed its expression under different conditions and genetic backgrounds. Transcription from
PalkS
was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. The
PalkS
−10 region was similar to the consensus described for promoters recognized by
Escherichia coli
RNA polymerase bound to the alternative sigma factor ς
S
, which directs the expression of many stationary-phase genes. Reporter strains containing
PalkS-lacZ
transcriptional fusions showed that
PalkS
promoter is very weakly expressed in a
Pseudomonas putida
strain bearing an inactivated allele of the gene coding for ς
S
,
rpoS
. When
PalkS
was transferred to
E. coli
, transcription started at the same site and expression was higher in stationary phase only if ς
S
-RNA polymerase was present. The low levels of AlkS protein generated in the absence of ς
S
were enough to support a partial induction of the
PalkB
promoter. The −10 and −35 regions of
PalkS
promoter also show some similarity to the consensus recognized by ς
D
-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase
PalkS
is probably recognized both by ς
D
-RNA polymerase (inefficiently) and by ς
S
-RNA polymerase (present at low levels), leading to low-level expression of the
alkS
gene. ς
S
-RNA polymerase would be responsible for the high level of activity of
PalkS
observed in stationary phase.
Uracil-Induced Down-Regulation of the Yeast Uracil Permease
Authors: Séron Karin , Blondel Marie-Odile , Haguenauer-Tsapis Rosine , Volland Christiane
Published: December 2019

In
Saccharomyces cerevisiae
the
FUR4
-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the
FUR4
mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.
Differential Dependence of Levansucrase and α-Amylase Secretion on SecA (Div) during the Exponential Phase of Growth of
Bacillus subtilis
Authors: Leloup Laurence , Driessen Arnold J. M. , Freudl Roland , Chambert Régis , Petit-Glatron Marie-Françoise
Published: December 2019

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a
degU32
(Hy)
Bacillus subtilis
strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and α-amylase overexpressed under the control of the
sacB
leader region. The level of SecA was reduced in the presence of sodium azide and in the
div341
thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing
secA
. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, α-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the α-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.
Characterization of the
ssnA
Gene, Which Is Involved in the Decline of Cell Viability at the Beginning of Stationary Phase in
Escherichia coli
Authors: Yamada Mamoru , Talukder Ali Azam , Nitta Takeshi
Published: December 2019

When grown in rich medium,
Escherichia coli
exhibits a drastic reduction of the number of viable cells at the beginning of stationary phase. The decline of cell viability was retarded by disruption of the
ssnA
gene, which was identified as a gene subject to RpoS-dependent negative regulation. Moreover,
ssnA
expression was induced at the time of decline of cell viability at early stationary phase. The viability decline was augmented in the
rpoS
background, and this augmentation was suppressed by
ssnA
mutation. Cloning of the
ssnA
gene in a multicopy plasmid, pBR322, caused small colony formation and slow growth in liquid medium. Cells harboring the
ssnA
clone showed aberrant morphology that included enlarged and filamentous shapes. The gene product was identified as a 44-kDa soluble protein, but its function could not be deduced by homology searching. From these results, we conclude that
ssnA
is expressed in response to a phase-specific signal(s) and that its expression level is controlled by RpoS, by a mechanism which may contribute to determination of cell number in the stationary phase.
Nucleoid-Independent Identification of Cell Division Sites in
Escherichia coli
Authors: Cook William R. , Rothfield Lawrence I.
Published: December 2019

The mechanism used by
Escherichia coli
to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.
Site-Directed Mutagenesis of Loop L3 of Sucrose Porin ScrY Leads to Changes in Substrate Selectivity
Authors: Ulmke Christine , Kreth Jens , Lengeler Joseph W. , Welte Wolfram , Schmid Kurt
Published: December 2019

The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores. We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB. In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport.
Sugar- and Nitrogen-Dependent Regulation of an
Amanita muscaria
Phenylalanine Ammonium Lyase Gene
Authors: Nehls U. , Ecke M. , Hampp R.
Published: December 2019

The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.
A Missense Mutation Accounts for the Defect in the Glycerol-3-Phosphate Acyltransferase Expressed in the
plsB26
Mutant
Authors: Heath Richard J. , Rock Charles O.
Published: December 2019

The
sn
-glycerol-3-phosphate acyltransferase (
plsB
) catalyzes the first step in membrane phospholipid formation. A conditional
Escherichia coli
mutant (
plsB26
) has a single missense mutation (G1045A) predicting the expression of an acyltransferase with an Ala349Thr substitution. The PlsB26 protein had a significantly reduced glycerol-3-phosphate acyltransferase specific activity coupled with an elevated

K
m

for glycerol-3-phosphate.
Expression of the
Methanobacterium thermoautotrophicum hpt
Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in
Escherichia coli
Authors: Sauer Jørgen , Nygaard Per
Published: December 2019

The
hpt
gene from the archaeon
Methanobacterium thermoautotrophicum
, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into
Escherichia coli
. The
hpt
-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine. The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the
hpt
gene. The recombinant enzyme was purified to apparent homogeneity.
Chromosome Methylation and Measurement of Faithful, Once and Only Once per Cell Cycle Chromosome Replication in
Caulobacter crescentus
Authors: Marczynski Gregory T.
Published: December 2019

Caulobacter crescentus
exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn
5
-based transposon, Tn
5
Ω-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn
5
Ω-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn
5
Ω-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn
5
Ω-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the “once and only once per cell cycle” rule of chromosome replication in
C. crescentus
. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.
acs1
of
Haemophilus influenzae
Type a Capsulation Locus Region II Encodes a Bifunctional Ribulose 5-Phosphate Reductase– CDP-Ribitol Pyrophosphorylase
Authors: Follens Anja , Veiga-da-Cunha Maria , Merckx Rita , van Schaftingen Emile , van Eldere Johan
Published: December 2019

The serotype-specific, 5.9-kb region II of the
Haemophilus influenzae
type a capsulation locus was sequenced and found to contain four open reading frames termed
acs1
to
acs4
. Acs1 was 96% identical to
H. influenzae
type b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107–118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis,
acs1
was cloned in an expression vector and overexpressed in
Escherichia coli
. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP
+
in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that
acs1
encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the
H. influenzae
type a capsular polysaccharide.
Role of Acid pH and Deficient Efflux of Pyrazinoic Acid in Unique Susceptibility of
Mycobacterium tuberculosis
to Pyrazinamide
Authors: Zhang Ying , Scorpio Angelo , Nikaido Hiroshi , Sun Zhonghe
Published: December 2019

Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against
Mycobacterium tuberculosis
in vitro except at an acidic pH.
M. tuberculosis
is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of
M. tuberculosis
are unknown. We found that in
M. tuberculosis
, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside
M. tuberculosis
cells. PZA-resistant
M. tuberculosis
complex organisms did not convert PZA into POA. Unlike
M. tuberculosis
, intrinsically PZA-resistant
M. smegmatis
converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of
M. tuberculosis
to PZA and that the natural PZA resistance of
M. smegmatis
is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.
Degradation of 1,2-Dibromoethane by
Mycobacterium
sp. Strain GP1
Authors: Poelarends Gerrit J. , van Hylckama Vlieg Johan E. T. , Marchesi Julian R. , Freitas Dos Santos Luisa M. , Janssen Dick B.
Published: December 2019

The newly isolated bacterial strain GP1 can utilize 1,2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaA
f
) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of
Rhodococcus rhodochrous
NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a
dhaA
gene region, which is also present in
R. rhodochrous
NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the
hheB
gene found in
Corynebacterium
sp. strain N-1074.
Disruption and Analysis of the
clpB
,
clpC
, and
clpE
Genes in
Lactococcus lactis
: ClpE, a New Clp Family in Gram-Positive Bacteria
Authors: Ingmer Hanne , Vogensen Finn K. , Hammer Karin , Kilstrup Mogens
Published: December 2019

In the genome of the gram-positive bacterium
Lactococcus lactis
MG1363, we have identified three genes (
clpC
,
clpE
, and
clpB
) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The
clpE
gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX
2
CX
22
CX
2
C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by
clpB
was strongly induced by heat shock and, while
clpC
mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in
clpB
,
clpC
, or
clpE
, we found that although the genes are part of the
L. lactis
heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins,
clpE
mutant cells formed smaller colonies than wild-type cells and
clpB
and
clpC
mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in
L. lactis
, could be necessary for degrading proteins generated by certain types of stress.
Iron Reductase for Magnetite Synthesis in the Magnetotactic Bacterium
Magnetospirillum magnetotacticum
Authors: Noguchi Yasushi , Fujiwara Taketomo , Yoshimatsu Katsuhiko , Fukumori Yoshihiro
Published: December 2019

Ferric iron reductase was purified from magnetotactic bacterium
Magnetospirillum
(formerly
Aquaspirillum
)
magnetotacticum
(ATCC 31632) to an electrophoretically homogeneous state. The enzyme was loosely bound on the cytoplasmic face of the cytoplasmic membrane and was found more frequently in magnetic cells than in nonmagnetic cells. The molecular mass of the purified enzyme was calculated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 36 kDa, almost the same as that calibrated by gel filtration analysis. The enzyme required NADH and flavin mononucleotide (FMN) as optimal electron donor and cofactor, respectively, and the activity was strongly inhibited by Zn
2+
acting as a partial mixed-type inhibitor. The

K
m

values for NADH and FMN were 4.3 and 0.035 μM, respectively, and the

K
i

values for Zn
2+
were 19.2 and 23.9 μM for NADH and FMN, respectively. When the bacterium was grown in the presence of ZnSO
4
, the magnetosome number in the cells and the ferric iron reductase activity declined in parallel with an increase in the ZnSO
4
concentration of the medium, suggesting that the ferric iron reductase purified in the present study may participate in magnetite synthesis.
A Periplasmic
d
-Alanyl-
d
-Alanine Dipeptidase in the Gram-Negative Bacterium
Salmonella enterica
Authors: Hilbert Friederike , del Portillo Francisco García , Groisman Eduardo A.
Published: December 2019

The VanX protein is a
d
-alanyl-
d
-alanine (
d
-Ala-
d
-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a
d
-Ala-
d
-Ala dipeptidase in the gram-negative species
Salmonella enterica
. The
Salmonella
enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes
d
-Ala-
d
-Ala,
dl
-Ala-
dl
-Phe, and
d
-Ala-Gly but not the tripeptides
d
-Ala-
d
-Ala-
d
-Ala and
dl
-Ala-
dl
-Lys-Gly or the dipeptides
l
-Ala-
l
-Ala,
N
-acetyl-
d
-Ala-
d
-Ala, and
l
-Leu-Pro. The
Salmonella
dipeptidase gene, designated
pcgL
, appears to have been acquired by horizontal gene transfer because
pcgL
-hybridizing sequences were not detected in related bacterial species and the G+C content of the
pcgL
-containing region (41%) is much lower than the overall G+C content of the
Salmonella
chromosome (52%). In contrast to wild-type
Salmonella
, a
pcgL
mutant was unable to use
d
-Ala-
d
-Ala as a sole carbon source. The
pcgL
gene conferred
d
-Ala-
d
-Ala dipeptidase activity upon
Escherichia coli
K-12 but did not allow growth on
d
-Ala-
d
-Ala. The PcgL protein localizes to the periplasmic space of
Salmonella
, suggesting that this dipeptidase participates in peptidoglycan metabolism.
Genes Coding for Phosphotransacetylase and Acetate Kinase in
Sinorhizobium meliloti
Are in an Operon That Is Inducible by Phosphate Stress and Controlled by PhoB
Authors: Summers Michael L. , Denton Michael C. , McDermott Timothy R.
Published: December 2019

Recent work in this laboratory has shown that the gene coding for acetate kinase (
ackA
) in
Sinorhizobium meliloti
is up-regulated in response to phosphate limitation. Characterization of the region surrounding
ackA
revealed that it is adjacent to
pta
, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (
orfA
),
pta
,
ackA
, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (
fabI
). Experiments combining enzyme assays, a chromosomal
lacZ
::
ackA
transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in
pta
and
ackA
all indicated that the
orfA-pta-ackA-fabI
genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of
orfA
. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in
S. meliloti
and to the consensus PHO box in
Escherichia coli
. Introduction of a
phoB
mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the
ackA
::
lacZ
fusion. Mutations in either
ackA
alone or both
pta
and
ackA
did not affect the nodulation or nitrogen fixation phenotype of
S. meliloti
.
Genetic Organization of the
Escherichia coli
K10 Capsule Gene Cluster: Identification and Characterization of Two Conserved Regions in Group III Capsule Gene Clusters Encoding Polysaccharide Transport Functions
Authors: Clarke Bradley R. , Pearce Rowan , Roberts Ian S.
Published: December 2019

Analysis of the
Escherichia coli
K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An
rfaH
mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS
3
element and a φR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes.
Cellular Locations of
Pseudomonas syringae
pv. syringae HrcC and HrcJ Proteins, Required for Harpin Secretion via the Type III Pathway
Authors: Deng Wen-Ling , Huang Hsiou-Chen
Published: December 2019

The complete
hrp-hrc-hrmA
cluster of
Pseudomonas syringae
pv. syringae 61 encodes 28 polypeptides. A saprophytic bacterium carrying this cluster is capable of secreting HrpZ—a harpin encoded by
hrpZ
—in an
hrp
-dependent manner, which suggests that this cluster contains sufficient components to assemble functional type III secretion machinery. Sequence data show that HrcJ and HrcC are putative outer membrane proteins, and nonpolar mutagenesis demonstrates they are all required for HrpZ secretion. In this study, we investigated the cellular localization of the HrcC and HrcJ proteins by Triton solubilization, sucrose-gradient isopycnic centrifugation, and immunogold labeling of the bacterial cell surface. Our results indicate that HrcC is indeed an outer membrane protein and that HrcJ is located between both membranes. Their membrane localization suggests that they might be involved in the formation of a supramolecular structure for protein secretion.
The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of thenirAGene and Characterization of the Recombinant Enzyme
Authors: Prudêncio Miguel , Eady Robert R. , Sawers Gary
Published: December 2019

ThenirAgene encoding the blue dissimilatory nitrite reductase fromAlcaligenes xylosoxidanshas been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. ThenirAgene was overexpressed inEscherichia coliand was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated fromA. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated fromA. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.
Specific Contacts between Residues in the DNA-Binding Domain of the TyrR Protein and Bases in the Operator of the
tyrP
Gene of
Escherichia coli
Authors: Hwang J. S. , Yang J. , Pittard A. J.
Published: December 2019

In the presence of tyrosine, the TyrR protein of
Escherichia coli
represses the expression of the
tyrP
gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051–1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the
tyrP
operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the
tyrP
operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the
tyrP
operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the ±5 positions in the TyrR boxes.
The ArgR Regulatory Protein, a Helper to the Anaerobic Regulator ANR during Transcriptional Activation of the
arcD
Promoter in
Pseudomonas aeruginosa
Authors: Lu Chung-Dar , Winteler Harald , Abdelal Ahmed , Haas Dieter
Published: December 2019

Pseudomonas aeruginosa
, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the
arcDABC
operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the
arcD
transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in
trans
, on the transcriptional regulator ArgR and, in
cis
, on an ArgR binding site centered at −73.5 bp in the
arcD
promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5′-TGACGC-3′, which deviated in only 1 base from the common sequence motif 5′-TGTCGC-3′ found in other ArgR binding sites of
P. aeruginosa
. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the
arc
operon, suggesting that ArgR alone does not activate the
arcD
promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of
arc
transcription.
Isolation of
Helicobacter pylori
Genes That Modulate Urease Activity
Authors: McGee David J. , May Carrie A. , Garner Rachel M. , Himpsl Janette M. , Mobley Harry L. T.
Published: December 2019

Helicobacter pylori
urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH
3
and CO
2
. We sought to identify
H. pylori
genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both
H. pylori
urease and the NixA nickel transporter.
Escherichia coli
SE5000 and DH5α transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An
H. pylori
DNA library was cotransformed into SE5000 (pHP8080) and DH5α (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained
hp0548
, a gene that potentially encodes a DNA helicase found within the
cag
pathogenicity island, and
hp0511
, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in
E. coli
(pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the
flbA
(also known as
flhA
) flagellar biosynthesis/regulatory gene (
hp1041
), an
lcrD
homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in
E. coli
(pHP8080) containing the subcloned
flbA
gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in
E. coli
(pHP8080) containing the
flbA
gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in
H. pylori
. These results suggest that
H. pylori
genes may modulate urease activity.
Accessory DNA in the Genomes of Representatives of the
Escherichia coli
Reference Collection
Authors: Hurtado Ana , Rodríguez-Valera Francisco
Published: December 2019

Different strains of the
Escherichia coli
reference collection (ECOR) differ widely in chromosomal size. To analyze the nature of the differential gene pool carried by different strains, we have followed an approach in which random amplified polymorphic DNA (RAPD) was used to generate several PCR fragments. Those present in some but not all the strains were screened by hybridization to assess their distribution throughout the ECOR collection. Thirteen fragments with various degrees of occurrence were sequenced. Three of them corresponded to RAPD markers of widespread distribution. Of these, two were housekeeping genes shown by hybridization to be present in all the
E. coli
strains and in
Salmonella enterica
LT2; the third fragment contained a paralogous copy of
dnaK
with widespread, but not global, distribution. The other 10 RAPD markers were found in only a few strains. However, hybridization results demonstrated that four of them were actually present in a large selection of the ECOR collection (between 42 and 97% of the strains); three of these fragments contained open reading frames associated with phages or plasmids known in
E. coli
K-12. The remaining six fragments were present in only between one and four strains; of these, four fragments showed no similarity to any sequence in the databases, and the other two had low but significant similarity to a protein involved in the
Klebsiella
capsule synthesis and to RNA helicases of archaeal genomes, respectively. Their percent GC, dinucleotide content, and codon adaptation index suggested an exogenous origin by horizontal transfer. These results can be interpreted as reflecting the presence of a large pool of strain-specific genes, whose origin could be outside the species boundaries.
Azorhizobium caulinodans
P
II
and GlnK Proteins Control Nitrogen Fixation and Ammonia Assimilation
Authors: Michel-Reydellet Nathalie , Kaminski P. Alexandre
Published: December 2019

We herein report that
Azorhizobium caulinodans
P
II
and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation. The disruption of both
glnB
and
glnK
resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state. P
II
and GlnK also controlled
nif
gene expression because NifA activated
nifH
transcription and nitrogenase activity was derepressed in
glnB glnK
double mutants, but not in wild-type bacteria, grown in the presence of ammonia.
Sequence Analysis of the Four Plasmid Genes Required To Produce the Circular Peptide Antibiotic Microcin J25
Authors: Solbiati José O. , Ciaccio Mirella , Farías Ricardo N. , González-Pastor José E. , Moreno Felipe , Salomón Raúl A.
Published: December 2019

A 4.8-kb plasmid region carrying the four genes
mcjABCD
necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced.
mcjA
encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of
mcjB
and
mcjC
are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.
The Global Nitrogen Regulator NtcA Regulates Transcription of the Signal Transducer P
II
(GlnB) and Influences Its Phosphorylation Level in Response to Nitrogen and Carbon Supplies in the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Lee Hyun-Mi , Vázquez-Bermúdez María Félix , de Marsac Nicole Tandeau
Published: December 2019

The P
II
protein is encoded by a unique
glnB
gene in
Synechococcus
sp. strain PCC 7942. Its expression has been analyzed in the wild type and in NtcA-null mutant cells grown under different conditions of nitrogen and carbon supply. RNA-DNA hybridization experiments revealed the presence of one transcript species 680 nucleotides long, whatever the nutrient conditions tested. A second transcript species, 620 nucleotides long, absent in the NtcA null mutant, was observed in wild-type cells that were nitrogen starved for 2 h under both high and low CO
2
and in the presence of nitrate under a high CO
2
concentration. Primer extension analysis indicated that the two transcript species are generated from two tandem promoters, a ς
70
Escherichia coli
-type promoter and an NtcA-dependent promoter, located 120 and 53 nucleotides, respectively, from the
glnB
initiation codon. The NtcA-dependent promoter is up-regulated under the conditions mentioned above, while the ς
70
E. coli
-type promoter displays constitutive levels of transcripts in the NtcA null mutant and slightly different levels in the wild-type cells, depending on the nitrogen and carbon supplies. In general, a good correlation between the amounts of the two transcript species and that of the P
II
protein was observed, as revealed by immunodetection with specific antibodies. The phosphorylation level of P
II
in the wild type is inversely correlated with nitrogen availability and directly correlated with higher CO
2
concentration. This regulation is correspondingly less stringent in the NtcA null mutant cells. In contrast, the dephosphorylation of P
II
is NtcA independent.
Membrane-Bound Division Proteins DivIB and DivIC of
Bacillus subtilis
Function Solely through Their External Domains in both Vegetative and Sporulation Division
Authors: Katis Vittorio L. , Wake R. Gerry
Published: December 2019

The
Bacillus subtilis
membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both
divIB
and
divIC
was performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of the
Escherichia coli
TolR protein.
B. subtilis
strains expressing the
divIB
and
divIC
hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed.
An Operon That Confers UV Resistance by Evoking the SOS Mutagenic Response in Streptococcal Conjugative Transposon Tn
5252
Authors: Munoz-Najar Ursula , Vijayakumar Moses N.
Published: December 2019

Streptococcus pneumoniae
Rx1 is capable of repairing lesions caused by DNA-damaging agents in an error-free manner but lacks a UV-inducible error-prone repair system due to the absence of chromosomally encoded UmuDC-like proteins. We have identified an operon-like structure 8 kb from the left end of the pneumococcal conjugative transposon Tn
5252
that confers SOS function in the host cells. DNA sequence analysis of this region revealed the presence of four open reading frames (ORFs). The deduced amino acid sequence of one of them, ORF13, which is capable of encoding a protein of 49.7 kDa, showed significant homology to UmuC, MucB, and other proteins involved in the SOS response. The carboxy-terminal region of another, ORF14, which is predicted to encode a 26-kDa polypeptide, shared similarity with UmuD- and MucA-like proteins that carry the amino acid residues recognized by the activated RecA* protein for proteolytic cleavage. The presence of plasmids carrying subcloned DNA from this region was found to restore UV-inducible mutagenic repair of chromosomal DNA in
Escherichia coli
cells defective in error-prone repair as well as in pneumococcus and
Enterococcus faecalis
UV202. Mutations within ORF13 abolished UV-induced mutagenesis but did not affect the conjugal transposition of the element.