bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of Low-Molecular-Weight Penicillin-Binding Proteins in
Bacillus subtilis
Spore Peptidoglycan Synthesis and Spore Properties
Authors: Popham David L. , Gilmore Meghan E. , Setlow Peter
Published: December 2019

The peptidoglycan cortex of endospores of
Bacillus
species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by
d
,
d
-carboxypeptidases. We report here the construction of mutant
B. subtilis
strains lacking all combinations of two and three of the four apparent
d
,
d
-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the
dacA
and
dacC
products have no significant role in spore peptidoglycan formation, the
dacB
and
dacF
products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a
dacB dacF
double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.
The
Azotobacter vinelandii
Response Regulator AlgR Is Essential for Cyst Formation
Authors: Núñez Cinthia , Moreno Soledad , Soberón-Chávez Gloria , Espín Guadalupe
Published: December 2019

Azotobacter vinelandii
produces the exopolysaccharide alginate, which is essential for the encystment process. In
Pseudomonas aeruginosa
, as well as in
A. vinelandii
, the ς
E
factor encoded by
algU
is required for transcription of
algD
, which encodes a key enzyme of the alginate biosynthetic pathway. The
P. aeruginosa
response regulator AlgR activates transcription of
algD
.
fimS
, located upstream
algR
, is proposed to encode the AlgR cognate sensor kinase. We have cloned and characterized the
A. vinelandii algR
gene; the deduced amino acid sequence of the protein encoded by this gene shows 79% identity with its
P. aeruginosa
homolog. Sequence analysis around the
algR
gene revealed the absence of a
fimS
homolog. Inactivation of
A. vinelandii algR
diminished alginate production by 50%, but did not affect
algD
transcription, and completely impaired the capacity to form mature cysts. Electron microscopy of the cyst structures formed by the
algR
mutant revealed that the encystment process is blocked at the step of exine formation. The transcriptional regulation of the
A. vinelandii algR
gene and the role of AlgR in alginate production differ significantly from those of its
P. aeruginosa
counterparts. These differences could be due to the fact that in
A. vinelandii
, alginate plays a role in encystment, a function not found in
P. aeruginosa
.
Protein Mobility in the Cytoplasm of
Escherichia coli
Authors: Elowitz Michael B. , Surette Michael G. , Wolf Pierre-Etienne , Stock Jeffry B. , Leibler Stanislas
Published: December 2019

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of
Escherichia coli
. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient,

D
a

, of GFP in
E. coli
DH5α was found to be 7.7 ± 2.5 μm
2
/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with

D
a

of 2.5 ± 0.6 μm
2
/s. In addition, GFP mobility can depend strongly on at least two factors: first,

D
a

is reduced to 3.6 ± 0.7 μm
2
/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces

D
a

to 4.0 ± 2.0 μm
2
/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of

D
a

reported here. These measurements have implications for the understanding of intracellular biochemical networks.
Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein
Authors: Lagos Rosalba , Villanueva Jorge E. , Monasterio Octavio
Published: December 2019

The gene coding for the immunity protein (
mceB
) and the structural gene of microcin E492 (
mceA
), a low-molecular-weight channel-forming bacteriocin produced by a strain of
Klebsiella pneumoniae
, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (
mceB
) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.
Isolation of a Putative
Candida albicans
Transcriptional Regulator Involved in Pleiotropic Drug Resistance by Functional Complementation of a
pdr1 pdr3
Mutation in
Saccharomyces cerevisiae
Authors: Talibi Driss , Raymond Martine
Published: December 2019

Three
Candida albicans
genes, designated
FCR
(for fluconazole resistance), have been isolated by their ability to complement the fluconazole (FCZ) hypersensitivity of a
Saccharomyces cerevisiae
mutant lacking the transcription factors Pdr1p and Pdr3p. Overexpression of any of the three
FCR
genes in the
pdr1 pdr3
mutant resulted in increased resistance of the cells to FCZ and cycloheximide and in increased expression of
PDR5
, a gene coding for a drug efflux transporter of the ATP-binding cassette superfamily and whose transcription is under the control of Pdr1p and Pdr3p. Deletion of
PDR5
in the
pdr1 pdr3
strain completely abrogated the ability of the three
FCR
genes to confer FCZ resistance, demonstrating that
PDR5
is required for
FCR
-mediated FCZ resistance in
S. cerevisiae
. The
FCR1
gene encodes a putative 517-amino-acid protein with an N-terminal Zn
2
C
6
-type zinc finger motif homologous to that found in fungal zinc cluster proteins, including
S. cerevisiae
Pdr1p and Pdr3p. We have constructed a
C. albicans
CAI4-derived mutant strain carrying a homozygous deletion of the
FCR1
gene and analyzed its ability to grow in the presence of FCZ. We found that the
fcr1Δ/fcr1
Δ mutant displays hyperresistance to FCZ and other antifungal drugs compared to the parental CAI4 strain. This hyperresistance could be reversed to wild-type levels by reintroduction of a plasmid-borne copy of
FCR1
into the
fcr1Δ/fcr1
Δ mutant. Taken together, our results indicate that the
FCR1
gene behaves as a negative regulator of drug resistance in
C. albicans
and constitute the first evidence that FCZ resistance can result from the inactivation of a regulatory factor such as Fcr1p.
The
Myxococcus xanthus pilQ
(
sglA
) Gene Encodes a Secretin Homolog Required for Type IV Pilus Biogenesis, Social Motility, and Development
Authors: Wall Daniel , Kolenbrander Paul E. , Kaiser Dale
Published: December 2019

The
Myxococcus xanthus sglA1
spontaneous mutation was originally isolated because it allowed dispersed cell growth in liquid yet retained the ability to form fruiting bodies. Consequently, most of today’s laboratory strains either contain the
sglA1
mutation or were derived from strains that carry it. Subsequent work showed that
sglA
was a gene for social gliding motility, a process which is mediated by type IV pili. Here
sglA
is shown to map to the major
pil
cluster and to encode a 901-amino-acid open reading frame (ORF) that is homologous to the secretin superfamily of proteins. Secretins form a channel in the outer membrane for the transport of macromolecules. The closest homologs found were PilQ proteins from
Pseudomonas aeruginosa
and
Neisseria gonorrhoeae
, which are required for type IV pili biogenesis and twitching motility. To signify these molecular and functional similarities, we have changed the name of
sglA
to
pilQ
. The hypomorphic
pilQ1
(
sglA1
) allele was sequenced and found to contain two missense mutations at residues 741 (G→S) and 762 (N→G). In addition, 19 independent social (S)-motility mutations are shown to map to the
pilQ
locus. In-frame deletions of
pilQ
and its downstream gene,
orfL
, were constructed.
pilQ
is shown to be essential for pilus biogenesis, S-motility, rippling, and fruiting body formation, while
orfL
is dispensable for these processes. The
pilQ1
allele, but not the Δ
pilQ
allele, was found to render cells hypersensitive to vancomycin, suggesting that PilQ1 alters the permeability properties of the outer membrane. Many differences between
pilQ1
and
pilQ
+
strains have been noted in the literature. We discuss some of these observations and how they may be rationalized in the context of our molecular and functional findings.
Activities of the
Porphyromonas gingivalis
PrtP Proteinase Determined by Construction of
prtP
-Deficient Mutants and Expression of the Gene in
Bacteroides
Species
Authors: Barkocy-Gallagher Genevieve A. , Foley Joseph W. , Lantz Marilyn S.
Published: December 2019

PrtP is a major cysteine proteinase of
Porphyromonas gingivalis
. The gene encoding this proteinase,
prtP
, was cloned into the
Escherichia coli-Bacteroides
shuttle vectors pFD288 and pFD340 and was expressed in
Bacteroides
cells, apparently under the control of its own promoter, when in pFD288, or a
Bacteroides
promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several
Bacteroides
species harboring the recombinant plasmids. The proteinase was recovered from
Bacteroides fragilis
ATCC 25285(pFD340-
prtP
) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by
Nα-p
-tosyl-
l
-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-
d
-Tyr-
l
-Pro-
l
-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither
B. fragilis
ATCC 25285(pFD340-
prtP
) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore,
P. gingivalis
W12, ATCC 33277, KDP110, and HG66 with knockout mutations in
prtP
were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other
P. gingivalis
proteins and describes the properties of
P. gingivalis
cells defective in the production of PrtP.
Adaptation of
Mycobacterium smegmatis
to Stationary Phase
Authors: Smeulders Marjan J. , Keer Jacquie , Speight Richard A. , Williams Huw D.
Published: December 2019

Mycobacterium tuberculosis
can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of
Mycobacterium smegmatis
as a model for mycobacterial persistence.
M. smegmatis
cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbon-limited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of
M. smegmatis
. These results argue against stationary-phase
M. smegmatis
cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.
Doxorubicin Overproduction in
Streptomyces peucetius
: Cloning and Characterization of the
dnrU
Ketoreductase and
dnrV
Genes and the
doxA
Cytochrome P-450 Hydroxylase Gene
Authors: Lomovskaya Natalie , Otten Sharee L. , Doi-Katayama Yukiko , Fonstein Leonid , Liu Xiao-Chun , Takatsu Toshio , Inventi-Solari Augusto , Filippini Silvia , Torti Francesca , Colombo Anna Luisa , Hutchinson C. Richard
Published: December 2019

Doxorubicin-overproducing strains of
Streptomyces peucetius
ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that contains
dpsH
, the
dpsG
polyketide synthase gene, the putative
dnrU
ketoreductase gene,
dnrV
, and the
doxA
cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacing
dnrU
,
dnrV
,
doxA
, or
dpsH
with mutant alleles and of
doxA
overexpression on the production of the principal anthracycline metabolites of
S. peucetius
were studied. The exact roles of
dpsH
and
dnrV
could not be established, although
dnrV
is implicated in the enzymatic reactions catalyzed by DoxA, but
dnrU
appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a
dnrX dnrU
(N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379–2386, 1998) double mutant and a
dnrX dnrU dnrH
(C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996) triple mutant. Overexpression of
doxA
in a
doxA::aphII
mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of
dnrV
and
doxA
jointly in the
dnrX dnrU
double mutant or the
dnrX dnrU dnrH
triple mutant increased the DXR titer 36 to 86%.
Convergent Pathways for Utilization of the Amino Sugars
N
-Acetylglucosamine,
N
-Acetylmannosamine, and
N
-Acetylneuraminic Acid by
Escherichia coli
Authors: Plumbridge Jacqueline , Vimr Eric
Published: December 2019

N
-Acetylglucosamine (GlcNAc) and
N
-acetylneuraminic acid (NANA) are good carbon sources for
Escherichia coli
K-12, whereas
N
-acetylmannosamine (ManNAc) is metabolized very slowly. The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation. ManNAc is transported by the
manXYZ
-encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P. This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the
nagBA
-encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway. Two independent mutations are necessary for good growth on ManNAc. One mutation maps to
mlc
, and mutations in this gene are known to enhance the expression of
manXYZ
. The second regulatory mutation was mapped to the
nanAT
operon, which encodes the NANA transporter and NANA lyase. The combined action of the
nanAT
gene products converts extracellular NANA to intracellular ManNAc. The second regulatory mutation defines an open reading frame (ORF), called
yhcK
, as the gene for the repressor of the
nan
operon (
nanR
). Mutations in the repressor enhance expression of the
nanAT
genes and, presumably, three distal, previously unidentified genes,
yhcJIH
. Expression of just one of these downstream ORFs,
yhcJ
, is necessary for growth on ManNAc in the presence of an
mlc
mutation. The
yhcJ
gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase (
nanE
). Another putative gene in the
nan
operon,
yhcI
, likely encodes ManNAc kinase (
nanK
), which should phosphorylate the ManNAc liberated from NANA by the NanA protein. Use of NANA as carbon source by
E. coli
also requires the
nagBA
gene products. The existence of a ManNAc kinase and epimerase within the
nan
operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P.
Multiple Small Heat Shock Proteins in Rhizobia
Authors: Münchbach Martin , Nocker Andreas , Narberhaus Franz
Published: December 2019

Seven genes coding for small heat shock proteins (sHsps) inBradyrhizobium japonicumhave been identified. They are organized in five operons that are coordinately regulated by ROSE, a negativelycis-acting DNA element. The deduced sHsps can be divided into two separate classes: class A, consisting of proteins that show similarity toEscherichia coliIbpA and IbpB, and class B, whose members display significant similarity to other sHsps from prokaryotes and eukaryotes. Two-dimensional gel electrophoresis and Edman sequencing revealed the presence of at least 12 sHsps inB. japonicum, indicating a remarkable abundance of sHsps in this organism. Three additional members of class A and two potentially novel heat shock proteins were identified on the basis of their amino termini. The presence of multiple sHsps was also demonstrated for a variety ofRhizobiumandBradyrhizobiumspecies by immunoblot analysis and two-dimensional gel electrophoresis. An extensive database survey revealed that, in contrast to the rhizobia, other bacteria contain maximally two sHsps whereas many plants have been reported to possess a sHsp superfamily.
Role of Quinolinate Phosphoribosyl Transferase in Degradation of Phthalate by
Burkholderia cepacia
DBO1
Authors: Chang Hung-Kuang , Zylstra Gerben J.
Published: December 2019

Two distinct regions of DNA encode the enzymes needed for phthalate degradation by
Burkholderia cepacia
DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD
+
was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading
B. cepacia
strains have two dissimilar genes for this enzyme, while non-phthalate-degrading
B. cepacia
strains have only a single gene. The sequenced gene was labeled
ophE
, due to the fact that it is specifically induced by phthalate as shown by
lacZ
gene fusions. Insertional knockout mutants lacking
ophE
grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives
B. cepacia
an advantage over other phthalate-degrading bacteria in the environment.
Diverse Oxygenations Catalyzed by Carbazole 1,9a-Dioxygenase from
Pseudomonas
sp. Strain CA10
Authors: Nojiri Hideaki , Nam Jeong-Won , Kosaka Mikiko , Morii Ken-Ichi , Takemura Tetsuo , Furihata Kazuo , Yamane Hisakazu , Omori Toshio
Published: December 2019

Carbazole 1,9a-dioxygenase (CARDO) from
Pseudomonas
sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-
p
-dioxin. It was revealed by gas chromatography-mass spectrometry and
1
H and
13
C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2′,3-trihydroxydiphenylmethane and 2,2′,3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the
cis
dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to
cis
-1,2-dihydroxy-1,2-dihydronaphthalene and
cis
-2,3-dihydroxy-2,3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation,
cis
dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO.
Identification of Genetic Determinants for the Hemolytic Activity of
Streptococcus agalactiae
by IS
S1
Transposition
Authors: Spellerberg Barbara , Pohl Barbara , Haase Gerhard , Martin Simone , Weber-Heynemann Josephine , Lütticken Rudolf
Published: December 2019

Streptococcus agalactiae
is a poorly transformable bacterium and studies of molecular mechanisms are difficult due to the limitations of genetic tools. Employing the novel pGh9:IS
S1
transposition vector we generated plasmid-based mutant libraries of
S. agalactiae
strains O90R and AC475 by random chromosomal integration. A screen for mutants with a nonhemolytic phenotype on sheep blood agar led to the identification of a genetic locus harboring several genes that are essential for the hemolytic function and pigment production of
S. agalactiae
. Nucleotide sequence analysis of nonhemolytic mutants revealed that four mutants had distinct insertion sites in a single genetic locus of 7 kb that was subsequently designated
cyl
. Eight different open reading frames were identified:
cylX
,
cylD
,
cylG
,
acpC
,
cylZ
,
cylA
,
cylB
, and
cylE
, coding for predicted proteins with molecular masses of 11, 33, 26, 11, 15, 35, 32, and 78 kDa, respectively. The deduced amino acid sequence of the protein encoded by
cylA
harbors a conserved ATP-binding cassette (ABC) motif, and the predicted proteins encoded by
cylA
and
cylB
have significant similarities to the nucleotide binding and transmembrane proteins of typical ABC transporter systems. Transcription analysis by reverse transcription-PCR suggests that
cylX
to
cylE
are part of an operon. The requirement of
acpC
and
cylZABE
for hemolysin production of
S. agalactiae
was confirmed either by targeted mutagenesis with the vector pGh5, complementation studies with pAT28, or analysis of insertion elements in naturally occurring nonhemolytic mutants.
Histidinol Phosphate Phosphatase, Catalyzing the Penultimate Step of the Histidine Biosynthesis Pathway, Is Encoded by
ytvP
(
hisJ
) in
Bacillus subtilis
Authors: le Coq Dominique , Fillinger Sabine , Aymerich Stéphane
Published: December 2019

The deduced product of the
Bacillus subtilis ytvP
gene is similar to that of
ORF13
, a gene of unknown function in the
Lactococcus lactis
histidine biosynthesis operon. A
B. subtilis ytvP
mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in
B. subtilis
was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the
ytvP
mutant, while purified glutathione
S
-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by
ytvP
in
B. subtilis
and led us to rename this gene
hisJ
. Together with the HolPase of
Saccharomyces cerevisiae
and the presumed HolPases of
L. lactis
and
Schizosaccharomyces pombe
, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of
Escherichia coli
,
Salmonella typhimurium
, and
Haemophilus influenzae
.
A Novel
Campylobacter jejuni
Two-Component Regulatory System Important for Temperature-Dependent Growth and Colonization
Authors: Brás Ana M. , Chatterjee Shimonti , Wren Brendan W. , Newell Diane G. , Ketley Julian M.
Published: December 2019

Campylobacter jejuni
colonizes the intestines of domestic and wild animals and is a common cause of human diarrheal disease. We identified a two-component regulatory system, designated the RacR-RacS (reduced ability to colonize) system, that is involved in a temperature-dependent signalling pathway. A mutation of the response regulator gene
racR
reduced the organism’s ability to colonize the chicken intestinal tract and resulted in temperature-dependent changes in its protein profile and growth characteristics.
NahY, a Catabolic Plasmid-Encoded Receptor Required for Chemotaxis of
Pseudomonas putida
to the Aromatic Hydrocarbon Naphthalene
Authors: Grimm Ann C. , Harwood Caroline S.
Published: December 2019

Pseudomonas putida
G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene,
nahY
, was found to be cotranscribed with
meta
cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The
nahY
gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A
P. putida
G7
nahY
mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of
nahY
on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.
Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from
Listeria monocytogenes
BM4293 among Gram-Positive and Gram-Negative Bacteria
Authors: Charpentier Emmanuelle , Gerbaud Guy , Courvalin Patrice
Published: December 2019

We determined the sequence and genetic organization of plasmid pIP823, which contains the
dfrD
gene;
dfrD
confers high-level trimethoprim resistance to
Listeria monocytogenes
BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The
rep
gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic
Bacillus
sp. and pUB110 from
Staphylococcus aureus
. The
mob
gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included
L. monocytogenes
,
Enterococcus faecalis
,
S. aureus
,
Bacillus subtilis
, and
Escherichia coli
. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between
L. monocytogenes
and
E. faecalis
, between
L. monocytogenes
and
E. coli
, and between strains of
E. coli
, and by the streptococcal conjugative transposon Tn
1545
from
L. monocytogenes
to
E. faecalis
, and from
L. monocytogenes
and
E. faecalis
to
E. coli
. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in
Listeria
spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.
Mapping an Interface of SecY (PrlA) and SecE (PrlG) by Using Synthetic Phenotypes and In Vivo Cross-Linking
Authors: Harris Chris R. , Silhavy Thomas J.
Published: December 2019

SecY and SecE are integral cytoplasmic membrane proteins that form an essential part of the protein translocation machinery in
Escherichia coli
. Sites of direct contact between these two proteins have been suggested by the allele-specific synthetic phenotypes exhibited by pairwise combinations of
prlA
and
prlG
signal sequence suppressor mutations in these genes. We have introduced cysteine residues within the first periplasmic loop of SecY and the second periplasmic loop of SecE, at a specific pair of positions identified by this genetic interaction. The expression of the cysteine mutant pair results in a dominant lethal phenotype that requires the presence of DsbA, which catalyzes the formation of disulfide bonds. A reducible SecY-SecE complex is also observed, demonstrating that these amino acids must be sufficiently proximal to form a disulfide bond. The use of cysteine-scanning mutagenesis enabled a second contact site to be discovered. Together, these two points of contact allow the modeling of a limited region of quaternary structure, establishing the first characterized site of interaction between these two proteins. This study proves that actual points of protein-protein contact can be identified by using synthetic phenotypes.
The Physiological Contribution of
Acinetobacter
PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK
Authors: D’Argenio David A. , Segura Ana , Coco Wayne M. , Bünz Patricia V. , Ornston L. Nicholas
Published: December 2019

VanK is the fourth member of the ubiquitous major facilitator superfamily of transport proteins to be identified that, together with PcaK, BenK, and MucK, contributes to aromatic catabolism in
Acinetobacter
sp. strain ADP1. VanK and PcaK have overlapping specificity for
p
-hydroxybenzoate and, most clearly, for protocatechuate: inactivation of both proteins severely impairs growth with protocatechuate, and the activity of either protein alone can mask the phenotype associated with inactivation of its homolog. Furthermore,
vanK pcaK
double-knockout mutants appear completely unable to grow in liquid culture with the hydroaromatic compound quinate, although such cells on plates convert quinate to protocatechuate, which then accumulates extracellularly and is readily visible as purple staining. This provides genetic evidence that quinate is converted to protocatechuate in the periplasm and is in line with the early argument that quinate catabolism should be physically separated from aromatic amino acid biosynthesis in the cytoplasm so as to avoid potential competition for intermediates common to both pathways. Previous studies of aromatic catabolism in
Acinetobacter
have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the β-ketoadipate pathway and preventing the toxic accumulation of carboxymuconate. By using this procedure, strains with a mutation in structural or regulatory genes blocking degradation of vanillate,
p
-hydroxybenzoate, or protocatechuate were selected. In this study, the overlapping specificity of the VanK and PcaK permeases was exploited to directly select strains with a mutation in either
vanK
or
pcaK
. Spontaneous mutations identified in
vanK
include a hot spot for frameshift mutation due to contraction of a G
6
mononucleotide repeat as well as point mutations producing amino acid substitutions useful for analysis of VanK structure and function. Preliminary second-site suppression analysis using transformation-facilitated PCR mutagenesis in one VanK mutant gave results similar to those using LacY, the prototypic member of the major facilitator superfamily, consistent with the two proteins having a similar mechanism of action. The selection for transport mutants described here for
Acinetobacter
may also be applicable to
Pseudomonas putida
, where the PcaK permease has an additional role in chemotaxis.
Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in
Magnaporthe grisea
, Is Inhibited by Self-Inhibitors and Requires Surface Attachment
Authors: Liu Zhi-Mei , Kolattukudy Pappachan E.
Published: December 2019

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of
Magnaporthe grisea
, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in
Colletotrichum gloeosporioides
and
Colletotrichum trifolii
differentiation. After calmodulin cDNA and genomic DNA from
M. grisea
were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the
M. grisea
genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of
M. grisea
appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.
Importance of a 5′ Stem-Loop for Longevity of
papA
mRNA in
Escherichia coli
Authors: Bricker Angela L. , Belasco Joel G.
Published: December 2019

High-level expression of the major pilus subunit (PapA) of uropathogenic strains of
Escherichia coli
results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of
papA
mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This
papA
RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the
papA
message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the
papA
5′ untranslated region, as evidenced by the significant acceleration in
papA
mRNA decay that results from its removal.
Analysis of the 5′ Portion of the Type 19A Capsule Locus Identifies Two Classes of
cpsC
,
cpsD
, and
cpsE
Genes in
Streptococcus pneumoniae
Authors: Morona Judy K. , Morona Renato , Paton James C.
Published: December 2019

Analysis of the sequence data obtained from the 5′ portion of the
Streptococcus pneumoniae
type 19A capsular polysaccharide biosynthesis locus (
cps19a
) revealed that the first seven genes are homologous to the first seven genes in the type 19F (
cps19f
) locus. The former genes were designated
cps19aA
to -
G
and were 70 to 90% identical to their
cps19f
counterparts. Southern hybridization analysis of the
cps
loci from various
S. pneumoniae
serotypes with probes specific for the
cps19aC
,
cps19aD
, and
cps19aE
genes indicated a hybridization pattern complementary to that previously reported for
cps19fC
,
cps19fD
, and
cps19fE
. That is, all serotypes tested contained high-stringency homologues of either the
cps19aC
to -
E
genes or the
cps19fC
to -
E
genes, but not both. On this basis
S. pneumoniae cps
loci can be divided into two distinct classes. Long-range PCR was used to amplify the
cps
regions between
cpsB
and
aliA
from a variety of pneumococcal serotypes. Direct sequencing of the 5′ end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of
cpsC
. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.
Salmonella typhimurium
IroN and FepA Proteins Mediate Uptake of Enterobactin but Differ in Their Specificity for Other Siderophores
Authors: Rabsch Wolfgang , Voigt Wolfgang , Reissbrodt Rolf , Tsolis Renée M. , Bäumler Andreas J.
Published: December 2019

Salmonella typhimurium
possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin. To determine whether both receptors have identical substrate specificities,
fepA
and
iroN
mutants and a double mutant were characterized. While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively.
A High Incidence of Prophage Carriage among Natural Isolates of
Streptococcus pneumoniae
Authors: Ramirez Mario , Severina Elena , Tomasz Alexander
Published: December 2019

The majority (591 of 791, or 76%) of
Streptococcus pneumoniae
clinical isolates examined showed the presence of two or more chromosomal
Sma
I fragments that hybridized with the
lytA
-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (
N
-acetylmuramic acid-
l
-alanine amidase). Strains carrying multiple copies of
lytA
homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several
lytA
-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the
lytA
gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the
lytA
-hybridizing fragments in excess of the host
lytA
represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of
lytA
indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.
Modes of Action of Five Different Endopectate Lyases fromErwinia chrysanthemi3937
Authors: Roy Caroline , Kester Harry , Visser Jaap , Shevchik Vladimir , Hugouvieux-Cotte-Pattat Nicole , Robert-Baudouy Jeanine , Benen Jacques
Published: December 2019

Five endopectate lyases from the phytopathogenic bacteriumErwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character. PelA, PelI, and PelL released oligogalacturonates of different sizes, whereas PelD and PelB released mostly unsaturated dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer and trimer, except for PelL, which degraded the primary products to unsaturated tetramer and pentamer in addition to unsaturated dimer and trimer. The bond cleavage frequencies on oligogalacturonates revealed differences in the modes of action of these enzymes that were commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI and PelL. For PelA, preferential products were dependent on the sizes of the oligogalacturonates. Whereas PelB and PelD displayed their highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the largest substrate used. The bond cleavage frequencies and reaction rates were used to estimate the number of subsites of each enzyme.
Importance of
cis
Determinants and Nitrogenase Activity in Regulated Stability of the
Klebsiella pneumoniae
Nitrogenase Structural Gene mRNA
Authors: Simon Holly M. , Gosink Mark M. , Roberts Gary P.
Published: December 2019

The
Klebsiella pneumoniae
nitrogen fixation (
nif
) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30°C). Addition of O
2
or fixed nitrogen or temperature increases to 37°C or more result in the dramatic destabilization of the
nif
mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of
nif
transcriptional regulation, was used to define
cis
determinants required for the regulated stability of the 5.2-kb
nifHDKTY
mRNA and to test the model suggested by earlier work that NifA is required in
trans
to stabilize
nif
mRNA under
nif
-derepressing conditions. O
2
regulation of
nifHDKTY
mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of
nifH
, indicating that a site(s) required for the O
2
-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing
nif
mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of
nifY
mutants and overexpression suggests a possible involvement of the protein in this sensing process.
Mutational Analysis of theStreptococcus pneumoniaeBimodular Class A Penicillin-Binding Proteins
Authors: Paik Johanna , Kern Iza , Lurz Rudi , Hakenbeck Regine
Published: December 2019

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the threeStreptococcus pneumoniaeclass A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disruptedpbp2agene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors inS. pneumoniae.
Characterization of a
dam
Mutant of
Serratia marcescens
and Nucleotide Sequence of the
dam
Region
Authors: Ostendorf Tammo , Cherepanov Peter , de Vries Johann , Wackernagel Wilfried
Published: December 2019

The DNA of
Serratia marcescens
has N
6
-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from
S. marcescens
Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (
dam
) coding for the adenine methyltransferase (Dam enzyme) of
S. marcescens
was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a
dam-13
::Tn
9
mutant of
Escherichia coli
. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of
E. coli
. The
dam
gene is located between flanking genes which are similar to those found to the sides of the
E. coli dam
gene. The results of complementation studies indicated that like Dam of
E. coli
and unlike Dam of
Vibrio cholerae
, the Dam enzyme of
S. marcescens
plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors.
The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple Bacterium
Rhodobacter capsulatus
Authors: Horken Kempton M. , Tabita F. Robert
Published: December 2019

Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism
Rhodobacter sphaeroides
, the form I RubisCO from
R. capsulatus
is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified the
R. capsulatus
form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to
R. capsulatus
. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes from
R. capsulatus
and
R. sphaeroides
, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of
R. capsulatus
form I RubisCO. The
R. capsulatus
form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO
2
, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO
2
. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO
2
affinity.