bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Characterization of SusG, an Outer Membrane Protein Essential for Starch Utilization by
Bacteroides thetaiotaomicron
Authors: Shipman Joseph A. , Cho Kyu Hong , Siegel Hilary A. , Salyers Abigail A.
Published: December 2019

Results from previous studies had suggested that
Bacteroides thetaiotaomicron
utilizes starch by binding the polysaccharide to the bacterial surface and subsequently degrading the polymer by using cell-associated enzymes. Most of the starch-degrading activity was localized to the periplasm, but a portion appeared to be membrane associated. This raised the possibility that some breakdown might occur in the outer membrane prior to exposure of the polysaccharide to the periplasmic polysaccharide-degrading enzymes. In this study, we show that SusG, an outer membrane protein which has been shown genetically to be essential for starch utilization, has enzymatic activity. Results of protease accessibility experiments support the hypothesis that SusG is exposed on the cell surface. Results of [
14
C]starch binding assays, however, show that SusG plays a negligible role in binding of starch to the cell surface. Consistent with this, SusG has a relatively high

K
m

for starch and by itself is not sufficient to allow cells to grow on starch or to bind starch. Hence, the main role of SusG is to hydrolyze starch, but the binding of starch to the cell surface is evidently mediated by other proteins presumably interacting with SusG.
Cloning and Analysis of the Capsid Morphogenesis Genes of
Pseudomonas aeruginosa
Bacteriophage D3: Another Example of Protein Chain Mail?
Authors: Gilakjan Zeynab A. , Kropinski Andrew M.
Published: December 2019

The terminal DNA restriction fragments (
Pst
I-D and -B) of
Pseudomonas aeruginosa
bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation (ρ = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor. This is identical to the situation observed with coliphage HK97.
Novel Organization of Genes Involved in Prophage Excision Identified in the Temperate Lactococcal Bacteriophage TP901-1
Authors: Breüner Anne , Brøndsted Lone , Hammer Karin
Published: December 2019

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (
orf7
) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1
attP
site which are integrated into
attB
on the chromosome of
Lactococcus lactis
. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the
upp
gene (encoding uracil phosphoribosyltransferase) was designed, since
upp
mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10
−5
per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.
Negative Regulation of the Gene for Fe-Containing Superoxide Dismutase by an Ni-Responsive Factor in
Streptomyces coelicolor
Authors: Chung Hye-Jung , Choi Jae-Hyun , Kim Eun-Ja , Cho You-Hee , Roe Jung-Hye
Published: December 2019

In
Streptomyces coelicolor
, transcription of the
sodF
genes, encoding Fe-containing superoxide dismutases, is negatively regulated by nickel. Gel mobility shift assays with
sodF1
promoter fragments and cell extracts from the A3(2) strain indicate the presence of a nickel-responsive DNA-binding protein, most likely a transcriptional repressor. The boundary for the Ni-responsive
cis
-acting region was identified both in vitro and vivo. Ni does not regulate the level of the putative repressor but only the binding competence of this protein.
Genome-Wide Transcriptional Analysis of Aerobic and Anaerobic Chemostat Cultures of
Saccharomyces cerevisiae
Authors: ter Linde J. J. M. , Liang H. , Davis R. W. , Steensma H. Y. , van Dijken J. P. , Pronk J. T.
Published: December 2019

The yeast
Saccharomyces cerevisiae
is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen. Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays. This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate. Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions. Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions.
Functional Domains Present in the Mycobacterial Hemagglutinin, HBHA
Authors: Delogu Giovanni , Brennan Michael J.
Published: December 2019

Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an α-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.
Genetic Transfer of Large DNA Inserts to Designated Loci of the
Bacillus subtilis
168 Genome
Authors: Itaya Mitsuhiro
Published: December 2019

It was found that contiguous DNA segments of up to 50 kb can be transferred between
Bacillus subtilis
genomes when a sufficient length of the flanking genomic region is provided for homologous recombination, although the efficiency of transfer was reduced as the insert size increased. Inserts were translocated to different loci, where appropriate integration sites were created.
The HPr(Ser) Kinase of
Streptococcus salivarius
: Purification, Properties, and Cloning of the
hprK
Gene
Authors: Brochu Denis , Vadeboncoeur Christian
Published: December 2019

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of
Streptococcus salivarius
ATCC 25975 was purified, and the encoding gene (
hprK
) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the
S. salivarius
enzyme showed 45% identity with the
Bacillus subtilis
enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted
hprK
gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg
2+
for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from
S. salivarius
is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its

K
m

s for HPr and ATP are 31 μM and 1 mM, respectively.
Neisseria gonorrhoeae
PilA Is an FtsY Homolog
Authors: Arvidson Cindy Grove , Powers Ted , Walter Peter , So Magdalene
Published: December 2019

The
pilA
gene of
Neisseria gonorrhoeae
was initially identified in a screen for transcriptional regulators of
pilE
, the expression locus for pilin, the major structural component of gonococcal pili. The predicted protein sequence for PilA has significant homology to two GTPases of the mammalian signal recognition particle (SRP), SRP54 and SRα. Homologs of SRP54 and SRα were subsequently identified in bacteria (Ffh and FtsY, respectively) and appear to form an SRP-like apparatus in prokaryotes. Of the two proteins, PilA is the most similar to FtsY (47% identical and 67% similar at the amino acid level). Like FtsY, PilA is essential for viability and hydrolyzes GTP. The similarities between PilA and the bacterial FtsY led us to ask whether PilA might function as the gonococcal FtsY. In this work, we show that overproduction of PilA in
Escherichia coli
leads to an accumulation of pre-β-lactamase, similar to previous observations with other bacterial SRP components. Low-level expression of
pilA
in an
ftsY
conditional mutant can complement the
ftsY
mutation and restore normal growth to this strain under nonpermissive conditions. In addition, purified PilA can replace FtsY in an in vitro translocation assay using purified
E. coli
SRP components. A PilA mutant that is severely affected in its GTPase activity cannot replace FtsY in vivo or in vitro. However, overexpression of the GTPase mutant leads to the accumulation of pre-β-lactamase, suggesting that the mutant protein may interact with the SRP apparatus to affect protein maturation. Taken together, these results show that the gonococcal PilA is an FtsY homolog and that the GTPase activity is necessary for its function.
Quorum Sensing in
Burkholderia cepacia
: Identification of the LuxRI Homologs CepRI
Authors: Lewenza Shawn , Conway Barbara , Greenberg E. P. , Sokol Pamela A.
Published: December 2019

Burkholderia cepacia
has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible
N
-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of
B. cepacia
K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a
luxR
homolog that was designated
cepR
. The flanking DNA region was used to clone the wild-type copy of
cepR
. Sequence analysis revealed the presence of
cepI
, a
luxI
homolog, located 727 bp upstream and divergently transcribed from
cepR
. A
lux
box-like sequence was identified upstream of
cepI
. CepR was 36% identical to
Pseudomonas aeruginosa
RhlR and 67% identical to SolR of
Ralstonia solanacearum
. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a
cepR
mutation restored parental levels of ornibactin and protease but not lipase. An
N
-acylhomoserine lactone was purified from culture fluids and identified as
N
-octanoylhomoserine lactone. K56-I2, a
cepI
mutant, was created and shown not to produce
N
-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for
cepIR
in the regulation of extracellular virulence factor production by
B. cepacia.
Sequence and Function of LuxU: a Two-Component Phosphorelay Protein That Regulates Quorum Sensing in
Vibrio harveyi
Authors: Freeman Jeremy A. , Bassler Bonnie L.
Published: December 2019

Vibrio harveyi
regulates the expression of bioluminescence (
lux
) in response to cell density, a phenomenon known as quorum sensing. In
V. harveyi
, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.
Expression and Study of Recombinant ExoM, a β1-4 Glucosyltransferase Involved in Succinoglycan Biosynthesis in
Sinorhizobium meliloti
Authors: Lellouch Annemarie C. , Geremia Roberto A.
Published: December 2019

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative β1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from
Sinorhizobium meliloti
. The open reading frame
exoM
was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter.
Escherichia coli
BL21(DE3)/pLysS containing
exoM
expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in
E. coli
as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region.
E. coli
membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an
S. meliloti
mutant strain which accumulates the ExoM substrate (Glcβ1-4Glcβ1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a β1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcβ1-4Glcβ1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.
The
Enterococcus faecalis pyr
Operon Is Regulated by Autogenous Transcriptional Attenuation at a Single Site in the 5′ Leader
Authors: Ghim Sa-Youl , Kim Charles C. , Bonner Eric R. , D’Elia John N. , Grabner Gail K. , Switzer Robert L.
Published: December 2019

The 5′ end of the
Enterococcus faecalis pyr
operon specifies, in order, the promoter, a 5′ untranslated leader, the
pyrR
gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the
pyrP
gene encoding a uracil permease, a 13-nt intercistronic region, and the
pyrB
gene encoding aspartate transcarbamylase. The 5′ leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the
Bacillus subtilis pyr
operon, are present in the
pyrR-pyrP
or
pyrP-pyrB
intercistronic regions. Several lines of evidence demonstrate that the
E. faecalis pyr
operon is repressed by uracil via transcriptional attenuation at the single 5′ leader termination site and that attenuation is mediated by the PyrR protein.
Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in
Schizosaccharomyces pombe
Authors: Tanaka Naotaka , Awai Atsuro , Bhuiyan M. Shah Alam , Fujita Kiyotaka , Fukui Hiroshi , Takegawa Kaoru
Published: December 2019

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the
gsf1
mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike
Saccharomyces cerevisiae FLO
mutants. The
gsf1
mutant coflocculated with
Schizosaccharomyces pombe
wild-type cells, while no coflocculation was found with galactose-deficient (
gms1
Δ) cells. Moreover, flocculation of the
gsf1
mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from
gms1
Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of
gsf1
-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in
S. pombe
.
Regulated Exopolysaccharide Production in
Myxococcus xanthus
Authors: Kim Sang-Hoon , Ramaswamy Srinivas , Downard John
Published: December 2019

Myxococcus xanthus
fibrils are cell surface-associated structures composed of roughly equal amounts of polysaccharide and protein. The level of
M. xanthus
polysaccharide production under different conditions in the wild type and in several mutants known to have alterations in fibril production was investigated. Wild-type exopolysaccharide increased significantly as cells entered the stationary phase of growth or upon addition of Ca
2+
to growing cells, and the polysaccharide-induced cells exhibited an enhanced capacity for cell-cell agglutination. The activity of the key gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase (Pck) also increased under these conditions. Most fibril-deficient mutants failed to produce polysaccharide in a stationary-phase- or Ca
2+
-dependent fashion. However, regulation of Pck activity was generally unimpaired in these mutant strains. In an
stk
mutant, which overproduces fibrils, polysaccharide production and Pck activity were constitutively high under the conditions tested. Polysaccharide production increased in most fibril-deficient strains when an
stk
mutant allele was present, indicating that these fibril-deficient mutants retained the basic cellular components required for fibril polysaccharide production. In contrast to other divalent cations tested, Sr
2+
effectively replaced Ca
2+
in stimulating polysaccharide production, and either Ca
2+
or Sr
2+
was required for fruiting-body formation by wild-type cells. By using transmission electron microscopy of freeze-substituted log-phase wild-type cells, fibril material was observed as a cell surface-associated layer of uniform thickness composed of filaments with an ordered structure.
The
Escherichia coli
Ada Protein Can Interact with Two Distinct Determinants in the ς
70
Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the
alkA
Promoter
Authors: Landini Paolo , Busby Stephen J. W.
Published: December 2019

The methylated form of the Ada protein (
me
Ada) activates transcription from the
Escherichia coli ada
,
aidB
, and
alkA
promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς
70
that affect Ada-activated transcription at
alkA
. Substitution to alanine of residues K593, K597, and R603 in ς
70
region 4 results in decreased Ada-dependent binding of RNA polymerase to the
alkA
promoter in vitro and impairs
alkA
transcription both in vivo and in vitro, suggesting that these residues define a determinant for
me
Ada-ς
70
interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307–13312, 1998), we showed that a set of negatively charged amino acids in ς
70
region 4 is involved in
me
Ada-ς
70
interaction at the
ada
and
aidB
promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect
me
Ada-dependent transcription at
ada
and
aidB
. Unlike the ς
70
amino acids involved in the interaction with
me
Ada at the
ada
and
aidB
promoters, K593, K597, and R603 are not conserved in ς
S
, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. While
me
Ada is able to promote transcription by the ς
S
form of RNA polymerase (Eς
S
) at
ada
and
aidB
, it fails to do so at
alkA
. We propose that
me
Ada can activate transcription at different promoters by contacting distinct determinants in ς
70
region 4 in a manner dependent on the location of the Ada binding site.
Roles of
Pseudomonas aeruginosa las
and
rhl
Quorum-Sensing Systems in Control of Twitching Motility
Authors: Glessner Alex , Smith Roger S. , Iglewski Barbara H. , Robinson Jayne B.
Published: December 2019

Pseudomonas aeruginosa
is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by
P. aeruginosa
is controlled through two quorum-sensing systems,
las
and
rhl
. We have obtained evidence that both the
las
and
rhl
quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112
c
ts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112
c
ts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of
las
and
rhl
mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of
P. aeruginosa
cells to adhere to human bronchial epithelial cells was also found to be dependent on the
rhl
quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.
Molecular Survey of the
Salmonella
Phage Typing System of Anderson
Authors: Schmieger Horst
Published: December 2019

Typing phages for
Salmonella
and the prophages of their typical propagation strains were analyzed at the DNA level. Most of them belong to the P22 branch of the lambdoid phages. Acquisition of new plating properties of the typing phages by propagation in particular strains can be due to different host specific modifications of the DNA or to recombination events with residing prophages which are reflected by changes in the respective DNA restriction patterns. It is concluded that the actually available set of typing phages is a historically unique combination of strains.
Functional Importance and Local Environments of the Cysteines in the Tetracycline Resistance Protein Encoded by Plasmid pBR322
Authors: Jewell Jean E. , Orwick Jill , Liu Jun , Miller Kurt W.
Published: December 2019

The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined. Cysteines are important but not essential for tetracycline transport activity. None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.
The Polar Flagellar Motor of
Vibrio cholerae
Is Driven by an Na
+
Motive Force
Authors: Kojima Seiji , Yamamoto Koichiro , Kawagishi Ikuro , Homma Michio
Published: December 2019

Vibrio cholerae
is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of
V. cholerae
. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na
+
-driven polar flagellar motor of
Vibrio alginolyticus
were found in the genome database of
V. cholerae
. The energy source of its flagellar motor was investigated. We examined the Na
+
dependence and the sensitivity to the Na
+
motor-specific inhibitor of the motility of the
V. cholerae
strains and present the evidence that the polar flagellar motor of
V. cholerae
is driven by an Na
+
motive force.
Construction and Analysis of Hybrid
Escherichia coli-Bacillus subtilis dnaK
Genes
Authors: Mogk Axel , Bukau Bernd , Lutz Rolf , Schumann Wolfgang
Published: December 2019

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since
Bacillus subtilis dnaK
was not able to complement an
Escherichia coli dnaK
null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the
B. subtilis
DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in
E. coli
. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in
E. coli
DnaK affected neither complementation of growth at high temperatures nor propagation of phage λ but abolished degradation of ς
32
.
A Novel Membrane Protein Influencing Cell Shape and Multicellular Swarming of
Proteus mirabilis
Authors: Hay Nicole A. , Tipper Donald J. , Gygi Daniel , Hughes Colin
Published: December 2019

Swarming in
Proteus mirabilis
is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called
ccmA
, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type
P. mirabilis
; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a
ccmA
null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the
ccmA
gene caused cells of both
Escherichia coli
and
P. mirabilis
to become enlarged and ellipsoidal.
Isolation of RNase H Genes That Are Essential for Growth of
Bacillus subtilis
168
Authors: Itaya Mitsuhiro , Omori Akira , Kanaya Shigenori , Crouch Robert J. , Tanaka Teruo , Kondo Kanae
Published: December 2019

Two genes encoding functional RNase H (EC
3.1.26.4
) were isolated from a gram-positive bacterium,
Bacillus subtilis
168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a
B. subtilis
DNA library. One (28.2 kDa) revealed high similarity to
Escherichia coli
RNase HII, encoded by the
rnhB
gene. The other (33.9 kDa) was designated
rnhC
and encodes
B. subtilis
RNase HIII. The
B. subtilis
genome has an
rnhA
homologue, the product of which has not yet shown RNase H activity. Analyses of all three
B. subtilis
genes revealed that
rnhB
and
rnhC
cannot be simultaneously inactivated. This observation indicated that in
B. subtilis
both the
rnhB
and
rnhC
products are involved in certain essential cellular processes that are different from those suggested by
E. coli rnh
mutation studies. Sequence conservation between the
rnhB
and
rnhC
genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single
rnhC
homologue in the small genome of
Mycoplasma
species.
Inducing Effect of Diamines on Transcription of the Cephamycin C Genes from the
lat
and
pcbAB
Promoters in
Nocardia lactamdurans
Authors: Leitão Ana Lucia , Enguita Francisco J. , De La Fuente Juan Luis , Liras Paloma , Martin Juan F.
Published: December 2019

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in
Nocardia lactamdurans
, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the
pcbAB
gene and to a lesser extent also the
lat
transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the
cefD
promoter was not affected. Coupling of the
lat
and
pcbAB
promoters to the reporter
xylE
gene showed that expression from the
lat
and
pcbAB
promoters was increased by addition of diaminopropane in
Streptomyces lividans
. Intracellular accumulation of diamines in
Nocardia
may be a signal to trigger antibiotic production.
Utilization of Electrically Reduced Neutral Red by
Actinobacillus succinogenes
: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation
Authors: Park D. H. , Zeikus J. G.
Published: December 2019

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from
Actinobacillus succinogenes
to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of
A. succinogenes
contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H
2
as the electron donor and on the fumarate concentration. During the growth of
Actinobacillus
on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(
n
-heptyl)-4-hydroxyquinoline
N
-oxide to whole cells or purified membranes inhibited succinate production from H
2
plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables
A. succinogenes
to utilize electricity as a significant source of metabolic reducing power.
Cloning of
fibA
, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure of
Peptostreptococcus micros
Authors: Kremer B. H. A. , Bijlsma J. J. E. , Kusters J. G. , de Graaff J. , van Steenbergen T. J. M.
Published: December 2019

Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogen
Peptostreptococcus micros
. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (Rg
Sm
) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the Rg
Sm
type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designated
fibA
, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of
fibA
exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.
Gliding Mutants of
Myxococcus xanthus
with High Reversal Frequencies and Small Displacements
Authors: Spormann Alfred M. , Kaiser Dale
Published: December 2019

Myxococcus xanthus
cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the
mgl
and
frz
genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in
mglAB
or in
cglB
, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min
−1
for Δ
mglAB
mutants and 2.7 min
−1
for
cglB
mutants, compared to 0.17 min
−1
for wild-type cells). The average gliding speed of Δ
mglAB
mutant cells was 40% of that of wild-type cells (on average 1.9 μm/min for Δ
mglAB
mutants, compared to 4.4 μm/min for wild-type cells). The
mglA
-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein:
mglB
mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min
−1
and an average speed of 2.6 μm/min. These values range between those exhibited by wild-type cells and by Δ
mglAB
mutant cells. Epistasis analysis of
frz
mutants, which are defective in aggregation and in single-cell reversals, showed that a
frzD
mutation, but not a
frzE
mutation, partially suppressed the
mglA
phenotype. In contrast to
mgl
mutants,
cglB
mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of
cglB
cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of
mglAB
cells was only partially reduced by a
pilR
mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore,
mglAB
mutants behave as if they were severely defective in A motility but only partially defective in S motility.
Localization of
Escherichia coli rpoC
Mutations That Affect RNA Polymerase Assembly and Activity at High Temperature
Authors: Nedea Eduard C. , Markov Dmitriy , Naryshkina Tatyana , Severinov Konstantin
Published: December 2019

We localized five
rpoC
(β′) mutations affecting
Escherichia coli
RNA polymerase assembly. The Ts4, XH56, and R120 mutations changed β′ residues conserved throughout eubacteria; the JE10092 mutation occurred in the hypervariable region;
rpoC1
(TsX) changed a universally conserved residue and corresponds to yeast
rpb1-1
. Thus, distinct, predominantly conserved β′ residues participate in interactions holding RNA polymerase together.
The
Escherichia coli
Homologue of Yeast Rer2, a Key Enzyme of Dolichol Synthesis, Is Essential for Carrier Lipid Formation in Bacterial Cell Wall Synthesis
Authors: Kato Jun-ichi , Fujisaki Shingo , Nakajima Ken-ichi , Nishimura Yukinobu , Sato Miyuki , Nakano Akihiko
Published: December 2019

We found in the
Escherichia coli
genome sequence a homologue of
RER2
, a
Saccharomyces cerevisiae
gene required for proper localization of an endoplasmic reticulum protein, and designated it
rth
(
RER2
homologue). The disruption of this gene was lethal for
E. coli
. To reveal its biological function, we isolated temperature-sensitive mutants of the
rth
gene. The mutant cells became swollen and burst at the nonpermissive temperature, indicating that their cell wall integrity was defective. Further analysis showed that the mutant cells were deficient in the activity of
cis
-prenyltransferase, namely, undecaprenyl diphosphate synthase, a key enzyme of the carrier lipid formation of peptidoglycan synthesis. The cellular level of undecaprenyl phosphate was in fact markedly decreased in the mutants. These results are consistent with the fact that the Rer2 homologue of
Micrococcus luteus
shows undecaprenyl diphosphate synthase activity (N. Shimizu, T. Koyama, and K. Ogura, J. Biol. Chem. 273:19476–19481, 1998) and demonstrate that
E. coli
Rth is indeed responsible for the maintenance of cell wall rigidity. Our work on the yeast
rer2
mutants shows that they are defective in the activity of
cis
-prenyltransferase, namely, dehydrodolichyl diphosphate synthase, a key enzyme of dolichol synthesis. Taking these data together, we conclude that the
RER2
gene family encodes
cis
-prenyltransferase, which plays an essential role in cell wall biosynthesis in bacteria and in dolichol synthesis in eukaryotic cells and has been well conserved during evolution.
Role of
CIS
in Replication of an IncB Plasmid
Authors: Praszkier J. , Pittard A. J.
Published: December 2019

Replication of the IncB plasmid pMU720 requires the synthesis of the
cis
-acting RepA protein and the presence of two DNA elements,
ori
and
CIS. CIS
is the 166-bp sequence separating the RepA coding sequence from
ori
. To investigate how this organization of the pMU720 replicon contributes to the mechanism of initiation of replication, mutations in the sequence and/or the length of
CIS
were introduced into the
CIS
region and their effects on the efficiency of replication of the pMU720 replicon in vivo was determined. The
CIS
region was found to be composed of two domains. The
repA
-proximal domain, which showed strong transcription termination activity, could be replaced by equivalent sequences from I-complex and IncL/M plasmids, whose replicons are organized in the same fashion as pMU720. Replacement by a
trpA
transcription terminator afforded only partial replication activity. The
repA
-distal domain was shown to be a spacer whose role was to position sequence(s) within
ori
on the correct face of the DNA helix vis-à-vis the
repA
-proximal portion of
CIS
. A model for the loading of RepA protein onto
ori
is discussed.
Adenosylcobalamin-Mediated Methyl Transfer by Toluate
cis
-Dihydrodiol Dehydrogenase of the TOL Plasmid pWW0
Authors: Lee Jang-Young , Park Hee-Sung , Kim Hak-Sung
Published: December 2019

We identified and characterized a methyl transfer activity of the toluate
cis
-dihydrodiol (4-methyl-3,5-cyclohexadiene-
cis
-1,2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene
cis
-dihydrodiol (3-methyl-4,5-cyclohexadiene-
cis
-1,2-diol). When the purified enzyme from the recombinant
Escherichia coli
containing the
xylL
gene was incubated with toluene
cis
-dihydrodiol in the presence of NAD
+
, the end products differed depending on the presence of adenosylcobalamin (coenzyme B
12
). The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor. Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin. Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction. The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation. To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well.
Disulfide Bridges Are Not Involved in Penicillin-Binding Protein 1b Dimerization in
Escherichia coli
Authors: Chalut Christian , Remy Marie-Hélène , Masson Jean-Michel
Published: December 2019

PBP1b can be found as a dimer in
Escherichia coli
. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers.
Earlier Positivity of Central-Venous- versus Peripheral-Blood Cultures Is Highly Predictive of Catheter-Related Sepsis
Authors: Blot François , Schmidt Eric , Nitenberg Gérard , Tancrède Cyrille , Leclercq Bernard , Laplanche Agnès , Andremont Antoine
Published: December 2019

To diagnose catheter-related sepsis without removing the catheter, we evaluated the differential positivity times of cultures of blood drawn simultaneously from central venous catheter and peripheral sites. In a 450-bed cancer reference center, simultaneous central- and peripheral-blood cultures were prospectively performed for patients with suspicion of catheter-related sepsis over an 18-month period. Data for 64 patients for whom the same microorganisms were found when central- and peripheral-blood samples were cultured were retrospectively reviewed by two independent physicians blinded to the differential positivity time values in order to establish or refute the diagnosis of catheter-related sepsis. The diagnosis was established in 28 cases, refuted in 14, and indeterminate in the remaining 22. The differential positivity time was significantly greater for patients with catheter-related sepsis (
P
< 10
−4
). A cutoff limit of +120 min had 100% specificity and 96.4% sensitivity for the diagnosis of catheter-related sepsis. These results strongly suggest that measurement of the differential positivity time might be a reliable tool facilitating the diagnosis of catheter-related sepsis in patients with an indwelling catheter.