bluuebunny/crossref_metadata_2025 huggingface dataset:
Legionella pneumophilaInvasion of MRC-5 Cells Induces Tyrosine Protein Phosphorylation
Authors: Susa Milorad , Marre Reinhard
Published: December 2019

After uptake and intracellular multiplication ofLegionella pneumophilain MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication ofL. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth ofL. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [35S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) ofLegionellaorigin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth ofL. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth ofL. pneumophila.
Invasive Ability of an
Escherichia coli
Strain Isolated from the Ileal Mucosa of a Patient with Crohn’s Disease
Authors: Boudeau Jerome , Glasser Anne-Lise , Masseret Estelle , Joly Bernard , Darfeuille-Michaud Arlette
Published: December 2019

Crohn’s disease (CD) is an inflammatory bowel disease in which
Escherichia coli
strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by
E. coli
strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic
E. coli
strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC)
E. coli
strains. Gentamicin protection assays showed that
E. coli
LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (
P
> 0.5) but significantly higher than that of ETEC (
P
< 0.03), EHEC (
P
< 0.005), EAggEC (
P
< 0.004) and DAEC (
P
< 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles
Salmonella
- or
Shigella
-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive
E. coli
strain and suggest the existence of a new potentially pathogenic group of
E. coli
, which we propose be designated adherent-invasive
E. coli
.
Resistance to Both Complement Activation and Phagocytosis in Type 3 Pneumococci Is Mediated by the Binding of Complement Regulatory Protein Factor H
Authors: Neeleman Chris , Geelen Sibyl P. M. , Aerts Piet C. , Daha Mohammed R. , Mollnes Tom E. , Roord John J. , Posthuma George , van Dijk Hans , Fleer André
Published: December 2019

To study the role of surface-associated proteins in the virulence ofStreptococcus pneumoniae, we used two serotype 3 strains, ATCC 6303 and WU2, and two PspA-negative mutants of WU2, an encapsulated one, JY1123 (Caps+/PspA−), and an unencapsulated one, DW3.8 (Caps−/PspA−). ATCC 6303 and WU2 were highly virulent in mice, while the virulence of JY1123 was slightly decreased (50% lethal doses [LD50s], 24, 6, and 147 CFU/mouse, respectively); DW3.8 was avirulent (LD50, 2 × 108CFU). In vitro, ATCC 6303, WU2, and JY1123 (Caps+/PspA−) strongly resisted complement activation and complement-dependent opsonophagocytosis, whereas DW3.8 (Caps−/PspA−) was easily phagocytized in fresh serum. Trypsin treatment of ATCC 6303, WU2, and JY1123 (Caps+/PspA−) resulted in enhanced complement activation and complement-dependent opsonophagocytosis. Trypsin had no deleterious effect on the polysaccharide capsule. In addition, trypsin pretreatment of ATCC 6303 strongly reduced virulence upon intraperitoneal challenge in mice. This indicated that surface proteins play a role in the resistance to complement activation and opsonophagocytosis and contribute to the virulence of type 3 pneumococci. In subsequent experiments, we could show that the modulation of complement activation was associated with surface components that bind complement regulator factor H; binding is trypsin sensitive and independent of prior complement activation. Immunoblotting of cell wall proteins of the virulent strain ATCC 6303 with anti-human factor H antibody revealed three factor H-binding proteins of 88, 150, and 196 kDa. Immunogold electron microscopy showed a close association of factor H-binding components with the outer surface of the cell wall. The role of these factor H-binding surface proteins in the virulence of pneumococci is interesting and warrants further investigation.
Replication-Defective Adenovirus Infection ReducesHelicobacter felisColonization in the Mouse in a Gamma Interferon- and Interleukin-12-Dependent Manner
Authors: Jiang Bo , Jordana Manel , Xing Zhou , Smaill Fionna , Snider Denis P. , Borojevic Rajka , Steele-Norwood Darlene , Hunt Richard H. , Croitoru Kenneth
Published: December 2019

Helicobacter infection leads to chronic inflammation of the stomach. Although the infection persists in spite of an immune response, animal studies have shown that adjuvant-based oral vaccines can protect against infection and even eliminate established infection. These vaccines are thought to induce a Th2 immune response, counterbalancing the Th1 response seen with natural infections. As a prelude to using adenovirus vectors carrying cytokine genes to modulate the immune response to establishedHelicobacter felisinfection, we first examined the effect of the replication-defective adenovirus (RDA) vector itself. C57BL/6 mice chronically infected withH. felis(8 to 10 weeks) received intramuscular injections of RDA. The effect of RDA on the severity ofH. feliscolonization and the degree of gastric inflammation was assessed 2 weeks later. RDA caused a significant decrease inH. feliscolonization without significantly altering the associated inflammation. RDA did not alter theH. felis-specific immunoglobulin G1 (IgG1), IgG2a, and IgA responses in the serum but was associated with an increase in gamma interferon (IFN-γ)-producing CD8+spleen cells. To determine if IFN-γ or Th1 cytokines were involved in the response to RDA, we examined RDA treatment ofH. felisinfection in mice lacking either IFN-γ or interleukin-12 (IL-12). RDA failed to alterH. feliscolonization in either of these two mouse strains. Thus, viral infection of mice chronically infected withH. felisled to a significant decrease inH. feliscolonization in an IFN-γ- and IL-12-dependent manner. These results demonstrate that Th1 responses associated with systemic viral infection can influence an establishedH. felisinfection.
Isolation of Peptides That Mimic Epitopes on a Malarial Antigen from Random Peptide Libraries Displayed on Phage
Authors: Adda Christopher G. , Tilley Leann , Anders Robin F. , Foley Michael
Published: December 2019

The ring-infected erythrocyte surface antigen (RESA) is a dense-granule protein of
Plasmodium falciparum
which binds to the cytoskeletal structure of the erythrocyte after parasite invasion. It is currently under trial as a vaccine candidate. In an effort to characterize further the antibody responses to this antigen, we have panned two independent libraries of random peptides expressed on the surface of filamentous phage with a monoclonal antibody (MAb 18/2) against RESA. One library consisted of a potentially constrained 17-mer peptide fused with the gpVIII phage coat protein, and the other displayed an unconstrained 15-mer as a fusion with the minor phage coat protein gpIII. Several rounds of biopanning resulted in enrichment from both libraries clones that interacted specifically with MAb 18/2 in protein-blotting and enzyme-linked immunosorbent assay experiments. Nucleotide sequencing of the random oligonucleotide insert revealed a common predominant motif: (S/T)AVDD. Several other clones had related but degenerate motifs. Thus, a monoclonal antibody against a malarial antigen can select common mimotopes from different random peptide libraries. We envisage many uses for this technology in malaria research.
Antiphosphorylcholine Antibody Levels Are Elevated in Humans with Periodontal Diseases
Authors: Schenkein Harvey A. , Gunsolley John C. , Best Al M. , Harrison M. Travis , Hahn Chin-Lo , Wu Jiuhua , Tew John G.
Published: December 2019

Human immunoglobulin G2 (IgG2) serum concentrations and the IgG2 antibody response toActinobacillus actinomycetemcomitanscan be influenced by genes, by environmental factors such as smoking, and by periodontal disease status. Examination of the IgG2 response to phosphorylcholine (PC), a response thought to be mainly induced by the C polysaccharide ofStreptococcus pneumoniae, suggested that periodontal disease status was also associated with this response. This prompted the hypothesis that PC is an important oral antigen associated with organisms in the periodontal flora and that anti-PC antibody is elevated as a consequence of periodontal disease. Subjects in various periodontal disease diagnostic categories in which attachment loss is exhibited were tested for anti-PC in serum. Those with adult periodontitis, localized juvenile periodontitis, generalized early-onset periodontitis, and gingival recession all had similar levels of anti-PC IgG2 serum antibody which were significantly greater than in the group of subjects with no attachment loss. Analysis of plaque samples from subgingival and supragingival sites in all diseases categories for reactivity with the anti-PC specific monoclonal antibody TEPC-15 revealed that a substantial proportion of the bacteria in dental plaque (30 to 40%) bear PC antigen; this antigen was not restricted to morphotypes resembling only cocci but was also present on rods and branched filamentous organisms. We found thatS. mitis,S. oralis, andS. sanguis, as well as oral actinomycetes, includingA. viscosus,A. odontolyticus, andA. israelii, incorporated substantial amounts of [3H]choline from culture media. Further analysis of antigens derived from these organisms by Western blot indicated thatS. oralis,S. sanguis,A. viscosus,A. odontolyticus, andA. israeliicontained TEPC-15-reactive antigens. The data show that many commonly occurring bacterial species found in dental plaque contain PC antigen and that immunization with plaque-derived PC antigens as a consequence of inflammation and periodontal attachment loss may influence systemic anti-PC antibody concentrations.
Role of Complement Receptors in Uptake ofMycobacterium aviumby Macrophages In Vivo: Evidence from Studies Using CD18-Deficient Mice
Authors: Bermudez Luiz E. , Goodman Joseph , Petrofsky Mary
Published: December 2019

Mycobacterium aviumis an intracellular pathogen that has been shown to invade macrophages by using complement receptors in vitro, but mycobacteria released from one cell can enter a second macrophage by using receptors different from complement receptors. Infection of CD18 (β2integrin) knockout mice and the C57 BL/6 control mice led to comparable levels of tissue infection at 1 day, 2 days, 1 week, and 3 weeks following administration of bacteria. A histopathological study revealed similar granulomatous lesions in the two mouse strains, with comparable numbers of organisms. In addition, transmission electron microscopy of spleen tissues from both strains of mice showed bacteria inside macrophages. Our in vivo findings support the hypothesis thatM. aviumin the host is likely to use receptors other than CR3 and CR4 receptors to enter macrophages with increased efficiency.
Molecular Mechanism for the Spontaneous Generation of Pigmentless
Porphyromonas gingivalis
Mutants
Authors: Chen Wen , Kuramitsu Howard K.
Published: December 2019

Porphyromonas gingivalis
is one of the pathogens associated with periodontal diseases, and its protease activity has been implicated as an important virulence factor. Kgp is the major Lys-gingipain protease of
P. gingivalis
and appears to be involved not only in enzyme activity but also in hemagglutination and the pigmented phenotype due to heme accumulation and/or hemoglobin binding. However, little information concerning the molecular mechanism for the spontaneous generation of pigmentless
P. gingivalis
mutants is currently available. In this study, several spontaneous pigmentless mutants of
P. gingivalis
were isolated and characterized. The results revealed that a portion of the
kgp
gene had been deleted from the chromosomes of the pigmentless mutants. This deletion appears to result from recombination between the highly homologous DNA sequences encoding the adhesin domains of the tandemly arranged
hagA
and
kgp
genes on the chromosomes of
P. gingivalis
strains.
Virulence ofMycobacterium tuberculosisCDC1551 and H37Rv in Rabbits Evaluated by Lurie’s Pulmonary Tubercle Count Method
Authors: Bishai William R. , Dannenberg Arthur M. , Parrish Nikki , Ruiz Rafael , Chen Ping , Zook Bernard C. , Johnson Walter , Boles James W. , Pitt M. Louise M.
Published: December 2019

The virulence of the CDC1551 strain ofMycobacterium tuberculosiswas compared to that of H37Rv in a rabbit inhalation model. While rabbits that inhaled the two strains produced equal numbers of grossly visible primary tubercles, CDC1551 tubercles were smaller and contained fewer bacilli than H37Rv tubercles. These findings suggest that a miniepidemic near the Kentucky-Tennessee border caused by CDC1551 was due not to increased virulence but to increased transmissibility.
Outer Membrane Proteins Omp10, Omp16, and Omp19 of
Brucella
spp. Are Lipoproteins
Authors: Tibor Anne , Decelle Béatrice , Letesson Jean-Jacques
Published: December 2019

The deduced sequences of the Omp10, Omp16, and Omp19 outer membrane proteins of
Brucella
spp. contain a potential bacterial lipoprotein processing sequence. After extraction with Triton X-114, these three proteins partitioned into the detergent phase. Processing of the three proteins is inhibited by globomycin, a specific inhibitor of lipoprotein signal peptidase. The three proteins were radioimmunoprecipitated from [
3
H]palmitic acid-labeled
Brucella abortus
lysates with monoclonal antibodies. These results demonstrate that Omp10, Omp16, and Omp19 are lipoproteins.
Metabolic Roles of a
Rhodobacter sphaeroides
Member of the ς
32
Family
Authors: Karls Russell K. , Brooks Jacqueline , Rossmeissl Peter , Luedke Janelle , Donohue Timothy J.
Published: December 2019

We report the role of a gene (
rpoH
) from the facultative phototroph
Rhodobacter sphaeroides
that encodes a protein (ς
37
) similar to
Escherichia coli
ς
32
and other members of the heat shock family of eubacterial sigma factors.
R. sphaeroides
ς
37
controls genes that function during environmental stress, since an
R. sphaeroides
ΔRpoH mutant is ∼30-fold more sensitive to the toxic oxyanion tellurite than wild-type cells. However, the ΔRpoH mutant lacks several phenotypes characteristic of
E. coli
cells lacking ς
32
. For example, an
R. sphaeroides
ΔRpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1, as well as its wild-type parent. In characterizing the response of
R. sphaeroides
to heat, we found that its growth temperature profile is different when cells generate energy by aerobic respiration, anaerobic respiration, or photosynthesis. However, growth of the ΔRpoH mutant is comparable to that of a wild-type strain under each of these conditions. The ΔRpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42°C, but it exhibited altered induction kinetics of ∼120-, 85-, 75-, and 65-kDa proteins. There was also reduced accumulation of several presumed heat shock transcripts (
rpoD
P
HS
,
groESL
1
, etc.) when aerobically grown ΔRpoH cells were placed at 42°C. Under aerobic conditions, it appears that another sigma factor enables the ΔRpoH mutant to mount a heat shock response, since either RNA polymerase preparations from an ΔRpoH mutant, reconstituted Eς
37
, or a holoenzyme containing a 38-kDa protein (ς
38
) each transcribed
E. coli
Eς
32
-dependent promoters. The lower growth temperature profile of photosynthetic cells is correlated with a difference in heat-inducible gene expression, since neither wild-type cells or the ΔRpoH mutant mount a typical heat shock response after such cultures were shifted from 30 to 37°C.
The Tryptophanase Gene Cluster of
Haemophilus influenzae
Type b: Evidence for Horizontal Gene Transfer
Authors: Martin Kimberly , Morlin Gregory , Smith Arnold , Nordyke Andrea , Eisenstark Abraham , Golomb Miriam
Published: December 2019

Among strains of
Haemophilus influenzae
, the ability to catabolize tryptophan (as detected by indole production) varies and is correlated with pathogenicity. Tryptophan catabolism is widespread (70 to 75%) among harmless respiratory isolates but is nearly universal (94 to 100%) among strains causing serious disease, including meningitis. As a first step in investigating the relationship between tryptophan catabolism and virulence, we have identified genes in pathogenic
H. influenzae
which are homologous to the tryptophanase (
tna
) operon of
Escherichia coli
. The
tna
genes are located on a 3.1-kb fragment between
nlpD
and
mutS
in the
H. influenzae
type b (Eagan) genome, are flanked by 43-bp direct repeats of an uptake signal sequence downstream from
nlpD
, and appear to have been inserted as a mobile unit within this sequence. The organization of this insertion is reminiscent of pathogenicity islands. The
tna
cluster is found at the same map location in all indole-positive strains of
H. influenzae
surveyed and is absent from reference type d and e genomes. In contrast to
H. influenzae
, most other
Haemophilus
species lack
tna
genes. Phylogenetic comparisons suggest that the
tna
cluster was acquired by intergeneric lateral transfer, either by
H. influenzae
or a recent ancestor, and that
E. coli
may have acquired its
tnaA
gene from a related source. Genomes of virulent
H. influenzae
resemble those of pathogenic enterics in having an island of laterally transferred DNA next to
mutS.
Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene (
cpe
) of
Clostridium perfringens
Authors: Zhao Yuling , Melville Stephen B.
Published: December 2019

Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of
Clostridium perfringens
enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the
cpe
promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of
cpe
, translational fusions containing the promoter deletions were made with the
gusA
gene of
Escherichia coli
, which codes for β-glucuronidase;
E. coli-C. perfringens
shuttle vectors carrying the fusions were introduced into
C. perfringens
by electroporation. In addition, in vitro transcription assays were performed with the
cpe
promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of
Bacillus subtilis
, the same compartment in which enterotoxin is synthesized in
C. perfringens.
AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes
Authors: Sadowsky Michael J. , Tong Zhaokun , de Souza Mervyn , Wackett Lawrence P.
Published: December 2019

Pseudomonas
sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding
N
-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway,
atzC
, was cloned from a
Pseudomonas
sp. strain ADP cosmid library as a 25-kb
Eco
RI DNA fragment in
Escherichia coli
. The
atzC
gene was further delimited by functional analysis following transposon Tn
5
mutagenesis and subcloned as a 2.0-kb
Eco
RI-
Ava
I fragment. An
E. coli
strain containing this DNA fragment expressed
N
-isopropylammelide isopropylamino hydrolase activity, metabolizing
N
-isopropylammelide stoichiometrically to cyanuric acid and
N
-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of
E. coli
cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.
Succinoglycan Production by
Rhizobium meliloti
Is Regulated through the ExoS-ChvI Two-Component Regulatory System
Authors: Cheng Hai-Ping , Walker Graham C.
Published: December 2019

The
Rhizobium meliloti exoS
gene is involved in regulating the production of succinoglycan, which plays a crucial role in the establishment of the symbiosis between
R. meliloti
Rm1021 and its host plant, alfalfa. The
exoS96
::Tn
5
mutation causes the upregulation of the succinoglycan biosynthetic genes, thereby resulting in the overproduction of succinoglycan. Through cloning and sequencing, we found that the
exoS
gene is a close homolog of the
Agrobacterium tumefaciens chvG
gene, which has been proposed to encode the sensor protein of the ChvG-ChvI two-component regulatory system, a member of the EnvZ-OmpR family. Further analyses revealed the existence of a newly discovered
A. tumefaciens chvI
homolog located just upstream of the
R. meliloti exoS
gene.
R. meliloti
ChvI may serve as the response regulator of ExoS in a two-component regulatory system. By using ExoS-specific antibodies, it was found that the ExoS protein cofractionated with membrane proteins, suggesting that it is located in the cytoplasmic membrane. By using the same antibodies, it was shown that the
exoS96
::Tn
5
allele encodes an N-terminal truncated derivative of ExoS. The cytoplasmic histidine kinase domain of ExoS was expressed in
Escherichia coli
and purified, as was the
R. meliloti
ChvI protein. The ChvI protein autophosphorylated in the presence of acetylphosphate, and the ExoS cytoplasmic domain fragment autophosphorylated at a histidine residue in the presence of ATP. The ChvI protein was phosphorylated in the presence of ATP only when the histidine kinase domain of ExoS was also present. We propose a model for regulation of succinoglycan production by
R. meliloti
through the ExoS-ChvI two-component regulatory system.
All Seven
comG
Open Reading Frames Are Required for DNA Binding during Transformation of Competent
Bacillus subtilis
Authors: Chung Y. S. , Dubnau D.
Published: December 2019

The seven proteins encoded by the
comG
operon of
Bacillus subtilis
exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in
comG
result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed.
Lagging-Strand Replication from the
ssoA
Origin of Plasmid pMV158 in
Streptococcus pneumoniae
: In Vivo and In Vitro Influences of Mutations in Two Conserved
ssoA
Regions
Authors: Kramer M. Gabriela , Khan Saleem A. , Espinosa Manuel
Published: December 2019

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin,
sso
. We have used the pMV158-derivative plasmid pLS1 (containing the
ssoA
type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the
ssoA
(the recombination site B [RS
B
] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in
Streptococcus pneumoniae
. Cells containing plasmids with mutations in the RS
B
accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS
B
region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.
Identification of an Na
+
-Dependent Malonate Transporter of
Malonomonas rubra
and Its Dependence on Two Separate Genes
Authors: Schaffitzel Christiane , Berg Michael , Dimroth Peter , Pos Klaas M.
Published: December 2019

Two membrane proteins encoded by the malonate fermentation gene cluster of
Malonomonas rubra
, MadL and MadM, have been synthesized in
Escherichia coli
. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na
+
dependent, and imposition of a ΔpNa
+
markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into
E. coli
BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate
−
is the transported malonate species. The stimulation of malonate uptake by Na
+
ions showed Michaelis-Menten kinetics, and a

K
m

for Na
+
of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na
+
/Hmalonate
−
symporter and that it is dependent on two separate genes.
Interactions between the Promoter Regions of Nitrogenase Structural Genes (
nifHDK2
) and DNA-Binding Proteins from N
2
- and Ammonium-Grown Cells of the Archaeon
Methanosarcina barkeri
227
Authors: Chien Yueh-tyng , Helmann John D. , Zinder Stephen H.
Published: December 2019

Transcription initiation in
Archaea
(archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of
nifHDK2
genes, encoding
Methanosarcina barkeri
nitrogenase, are present in N
2
-grown cells but not in ammonium-grown cells, indicating that
nif
transcription is regulated by the nitrogen source. In this study, we detected proteins in
M. barkeri
cell extracts that bind specifically to DNA containing the putative promoter region of
nifHDK2
. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (
mcr
) promoter region DNA and the
nifH2
promoter region DNA competed for a common factor(s). There was no binding to the
nifH2
promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for
mcr
genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the
nif
promoter region by extracts of N
2
-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N
2
-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of
nif
gene expression in
M. barkeri
in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.
Cloning and Physical Mapping of the
Eco
RI Fragments of the Giant Linear Plasmid SCP1
Authors: Redenbach Matthias , Ikeda Kazuya , Yamasaki Masayuki , Kinashi Haruyasu
Published: December 2019

A cosmid library was constructed for the 350-kb giant linear plasmid SCP1 and aligned on a successive linear map. Only a 0.8-kb gap has remained uncloned in the terminal inverted repeats close to both ends. Partial digestion of the aligned cosmids with
Eco
RI and hybridization with the flanking fragments of the vector enabled physical mapping of all of the
Eco
RI fragments. On this map, the methylenomycin biosynthetic gene cluster, the insertion sequence IS
466
, and the
sapCDE
genes coding for spore-associated proteins were localized.
Nonnative Proteins Induce Expression of theBacillus subtilisCIRCE Regulon
Authors: Mogk Axel , Völker Andrea , Engelmann Susanne , Hecker Michael , Schumann Wolfgang , Völker Uwe
Published: December 2019

The chaperone-encodinggroESLanddnaKoperons constitute the CIRCE regulon ofBacillus subtilis. Both operons are under negative control of the repressor protein HrcA, which interacts with the CIRCE operator and whose activity is modulated by the GroESL chaperone machine. In this report, we demonstrate that induction of the CIRCE regulon can also be accomplished by ethanol stress and puromycin. Introduction of thehrcAgene and a transcriptional fusion under the control of the CIRCE operator intoEscherichia coliallowed induction of this fusion by heat shock, ethanol stress, and overproduction of GroESL substrates. The expression level of thishrcA-bgaBfusion inversely correlated with the amount of GroE machinery present in the cells. Therefore, all inducing conditions seem to lead to induction via titration of the GroE chaperonins by the increased level of nonnative proteins formed. Puromycin treatment failed to induce the ςB-dependent general stress regulon, indicating that nonnative proteins in general do not trigger this response. Reconstitution of HrcA-dependent heat shock regulation ofB. subtilisinE. coliand complementation ofE. coli groESLmutants byB. subtilis groESLindicate that the GroE chaperonin systems of the two bacterial species are functionally exchangeable.
Role of the Multidrug Efflux Systems of
Pseudomonas aeruginosa
in Organic Solvent Tolerance
Authors: Li Xian-Zhi , Zhang Li , Poole Keith
Published: December 2019

Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in
Pseudomonas aeruginosa
. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as
n
-hexane and
p
-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of
P. aeruginosa
. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of
P. aeruginosa
to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.
Determining the Optimal Thymidine Concentration for Growing Thy
−
Escherichia coli
Strains
Authors: Molina Felipe , Jiménez-Sánchez Alfonso , Guzmán Elena C.
Published: December 2019

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy
−
strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy
−
Escherichia coli
strain. Using this method, we found that the
E. coli
“wild-type” strain MG1655 has a partial Thy
−
phenotype.
Multidrug Resistance following Expression of the
Escherichia coli marA
Gene in
Mycobacterium smegmatis
Authors: McDermott Patrick F. , White David G. , Podglajen Isabelle , Alekshun Michael N. , Levy Stuart B.
Published: December 2019

Expression of the
Escherichia coli
multiple antibiotic resistance
marA
gene cloned in
Mycobacterium smegmatis
produced increased resistance to multiple antimicrobial agents, including rifampin, isoniazid, ethambutol, tetracycline, and chloramphenicol. Cloned
marR
or
marA
cloned in the antisense direction had no effect. Resistance changes were lost with spontaneous loss of the plasmid bearing
marA
. A MarA mutant protein, having an insertional mutation within either of its two alpha-helices of the first putative helix-turn-helix domain, failed to produce the multiresistance phenotype in
E. coli
and
M. smegmatis
, indicating that this region is critical for MarA function. These results strongly suggest that
E. coli marA
functions in
M. smegmatis
and that a
mar
-like regulatory system exists in this organism.
Acyl Coenzyme A Synthetase from
Pseudomonas fragi
Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates
Authors: Fontes Rui , Günther Sillero Maria A. , Sillero Antonio
Published: December 2019

Acyl coenzyme A (CoA) synthetase (EC
6.2.1.8
) from
Pseudomonas fragi
catalyzes the synthesis of adenosine 5′-tetraphosphate (p
4
A) and adenosine 5′-pentaphosphate (p
5
A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-
O
-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap
4
A), and adenosine(5′)pentaphospho(5′)adenosine (Ap
5
A) are also substrates of the reaction yielding p
4
(d)A in the presence of tripolyphosphate (P
3
). UTP, CTP, and AMP are not substrates of the reaction. The

K
m

values for ATP and P
3
are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl
2
or CoCl
2
equimolecular with the sum of ATP and P
3
. The relative rates of synthesis of p
4
A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p
4
A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p
4
A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap
4
A (from ATP), Ap
5
A (from p
4
A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap
4
N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).
Characterization of thehcnABCGene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol AgentPseudomonas fluorescensCHA0
Authors: Laville Jacques , Blumer Caroline , Von Schroetter Christine , Gaia Valeria , Défago Geneviève , Keel Christoph , Haas Dieter
Published: December 2019

The secondary metabolite hydrogen cyanide (HCN) is produced byPseudomonas fluorescensfrom glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis inP. fluorescensCHA0 was investigated. The contiguous structural geneshcnABCencoding HCN synthase were expressed from the T7 promoter inEscherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of thehcnABCgenes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. ThehcnApromoter was mapped by primer extension; the −40 sequence (TTGGC … .ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT … .ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned fromP. fluorescensCHA0 and sequenced. ANR of strain CHA0 was most similar to ANR ofP. aeruginosaand CydR ofAzotobacter vinelandii. Ananrmutant ofP. fluorescens(CHA21) produced little HCN and was unable to express anhcnA-lacZtranslational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of thehcnA-lacZfusion. Mutant CHA21 as well as anhcndeletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused byThielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where theanrmutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.
An Autonomously Replicating Transforming Vector for
Sulfolobus solfataricus
Authors: Cannio Raffaele , Contursi Patrizia , Rossi Mosè , Bartolucci Simonetta
Published: December 2019

A plasmid able to transform and to be stably maintained both in
Sulfolobus solfataricus
and in
Escherichia coli
was constructed by insertion into an
E. coli
plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the
hph
(hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in
Sulfolobus
was increased by exposure of the cells to mitomycin C.
Appropriate Expression of Filamentous Phage f1 DNA Replication Genes II and X Requires RNase E-Dependent Processing and Separate mRNAs
Authors: Kokoska Robert J. , Steege Deborah A.
Published: December 2019

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected
Escherichia coli
hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on
rne
, the gene encoding the only
E. coli
endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.
Establishment of Prespore-Specific Gene Expression in
Bacillus subtilis
: Localization of SpoIIE Phosphatase and Initiation of Compartment-Specific Proteolysis
Authors: Lewis Peter J. , Wu Ling Juan , Errington Jeffery
Published: December 2019

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in
Bacillus subtilis
. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor ς
E
and the other dependent on the prespore-specific sigma factor ς
F
.
A Complex Pattern of Traveling Stripes Is Produced by Swimming Cells of
Bacillus subtilis
Authors: Mendelson Neil H. , Lega Joceline
Published: December 2019

Motile cells of
Bacillus subtilis
inadvertently escaped from the surface of an agar disk that was surrounded by a fluid growth medium and formed a migrating population in the fluid. When viewed from above, the population appeared as a cloud advancing unidirectionally into the fresh medium. The cell population became spontaneously organized into a series of stripes in a region behind the advancing cloud front. The number of stripes increased progressively until a saturation value of stripe density per unit area was reached. New stripes arose at a fixed distance behind the cloud front and also between stripes. The spacing between stripes underwent changes with time as stripes migrated towards and away from the cloud front. The global pattern appeared to be stretched by the advancing cloud front. At a time corresponding to approximately two cell doublings after pattern formation, the pattern decayed, suggesting that there is a maximum number of cells that can be maintained within the pattern. Stripes appear to consist of high concentrations of cells organized in sinking columns that are part of a bioconvection system. Their behavior reveals an interplay between bacterial swimming, bioconvection-driven fluid motion, and cell concentration. A mathematical model that reproduces the development and dynamics of the stripe pattern has been developed.
Purification and Properties of the F
1
F
o
ATPase of
Ilyobacter tartaricus
, a Sodium Ion Pump
Authors: Neumann Sandra , Matthey Ulrich , Kaim Georg , Dimroth Peter
Published: December 2019

The ATPase of
Ilyobacter tartaricus
was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F
1
F
o
ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, ɛ, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na
+
-translocating ATPase of
Propionigenium modestum
. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na
+
ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na
+
or Li
+
ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na
+
, Li
+
, or H
+
. Proton transport was specifically inhibited by Na
+
or Li
+
ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the
I. tartaricus
ATPase as a new member of the family of FS-ATPases, which use Na
+
as the physiological coupling ion for ATP synthesis.