bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonribosomal Peptide Synthesis and Toxigenicity of Cyanobacteria
Authors: Neilan Brett A. , Dittmann Elke , Rouhiainen Leo , Bass R. Amanda , Schaub Verena , Sivonen Kaarina , Börner Thomas
Published: December 2019

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546–1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69–72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.
Identification of a Putative P-Transporter Operon in the Genome of aBurkholderiaStrain Living inside the Arbuscular Mycorrhizal FungusGigaspora margarita
Authors: Ruiz-Lozano J. M. , Bonfante P.
Published: December 2019

This article reports the identification of a putative P-transporter operon in the genome of aBurkholderiasp. living in the cytoplasm of the arbuscular mycorrhizal fungusGigaspora margarita. Its presence suggests thatBurkholderiasp. has the potential for P uptake from this environment. This finding raises new questions concerning the importance of intracellular bacteria for mycorrhizal symbiosis.
The
pvc
Gene Cluster of
Pseudomonas aeruginosa
: Role in Synthesis of the Pyoverdine Chromophore and Regulation by PtxR and PvdS
Authors: Stintzi Alain , Johnson Zaiga , Stonehouse Martin , Ochsner Urs , Meyer Jean-Marie , Vasil Michael L. , Poole Keith
Published: December 2019

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the
Pseudomonas aeruginosa
siderophore pyoverdine, dubbed
pvcABCD
(where
pvc
stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the
pvc
genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore.
pvcABCD
expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.
First Chromosomal Restriction Map ofActinobacillus pleuropneumoniaeand Localization of Putative Virulence-Associated Genes
Authors: Oswald Winfried , Konine Denis V. , Rohde Judith , Gerlach Gerald-F.
Published: December 2019

Combined physical and genetic maps of the genomes ofActinobacillus pleuropneumoniaeAP76 (serotype 7 clinical isolate) and ofA. pleuropneumoniaeATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleasesApaI,AscI,NotI, andSalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between theApaI,AscI,NotI, andSalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The singleSalI site within the chromosome of strainA. pleuropneumoniaeAP76 was defined as position 1 of the map; for the map ofA. pleuropneumoniaeATCC 27088, the correspondingSalI site was chosen. Putative virulence-associated genes (apx,omlA,sodA,tbpBA,ureC, and a repeat element) and housekeeping genes (glyA,metJ,recA, andrhoAP) were positioned on the physical maps and located on theApaI andNotI fragments ofA. pleuropneumoniaeserotype reference strains.
Visualization of AqpZ-Mediated Water Permeability in
Escherichia coli
by Cryoelectron Microscopy
Authors: Delamarche Christian , Thomas Daniel , Rolland Jean-Paul , Froger Alexandrine , Gouranton Jean , Svelto Maria , Agre Peter , Calamita Giuseppe
Published: December 2019

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene,
aqpZ
, in
Escherichia coli
. By employing cryoelectron microscopy to compare
E. coli
cells containing (AqpZ
+
) and lacking (AqpZ
−
) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.
The Unique Chaperone Operon of
Thermotoga maritima
: Cloning and Initial Characterization of a Functional Hsp70 and Small Heat Shock Protein
Authors: Michelini Edward T. , Flynn Gregory C.
Published: December 2019

The hyperthermophilic eubacterium
Thermotoga maritima
possesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of
T. maritima
, one of the deepest-branching eubacteria known.
A Mycobacterial Extracytoplasmic Sigma Factor Involved in Survival following Heat Shock and Oxidative Stress
Authors: Fernandes Norvin D. , Wu Qi-long , Kong Dequan , Puyang Xiaoling , Garg Sumeet , Husson Robert N.
Published: December 2019

Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor,
sigH
, was cloned from
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including
M. fortuitum
and
M. avium
. While the
M. tuberculosis
and
M. smegmatis sigH
genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified in
M. smegmatis
and one of two identified in
M. bovis
BCG were found to have −35 promoter sequences that match the ECF-dependent −35 promoter consensus. Expression from these promoters was strongly induced by 50°C heat shock. In comparison to the wild type, an
M. smegmatis sigH
mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an
M. smegmatis sigH sigE
double mutant was found to be markedly decreased following 53°C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the
sigH
operon is required for complementation of the
sigH
stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.
The Chemotactic Response of
Vibrio anguillarum
to Fish Intestinal Mucus Is Mediated by a Combination of Multiple Mucus Components
Authors: O’Toole Ronan , Lundberg Susanne , Fredriksson Sten-Åke , Jansson Anita , Nilsson Bo , Wolf-Watz Hans
Published: December 2019

Chemotactic motility has previously been shown to be essential for the virulence of
Vibrio anguillarum
in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that
V. anguillarum
swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for
V. anguillarum
. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human
Vibrio
pathogen were made. A
cheR
mutant of a
Vibrio cholerae
El Tor strain was constructed, and it was found that
V. cholerae
and
V. anguillarum
exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.
A Region of ς
K
Involved in Promoter Activation by GerE in
Bacillus subtilis
Authors: Wade Kathryn H. , Schyns Ghislain , Opdyke Jason A. , Moran Charles P.
Published: December 2019

During endospore formation in
Bacillus subtilis
, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing ς
K
. GerE binds to a site on one of these promoters,
cotX
, that overlaps its −35 region. We tested the model that GerE interacts with ς
K
at the
cotX
promoter by seeking amino acid substitutions in ς
K
that interfered with GerE-dependent activation of the
cotX
promoter but which did not affect utilization of the ς
K
-dependent, GerE-independent promoter
gerE
. We identified two amino acid substitutions in ς
K
, E216K and H225Y, that decrease
cotX
promoter utilization but do not affect
gerE
promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in ς
K
on transcription in vitro. We found that these substitutions specifically reduced utilization of the
cotX
promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent
cotX
promoter activity, that the histidine at position 225 of ς
K
may interact with GerE at the
cotX
promoter, and that this interaction may facilitate the initial binding of ς
K
RNA polymerase to the
cotX
promoter. We also found that the alanine substitutions at positions 216 and 225 of ς
K
had no effect on utilization of the GerE-dependent promoter
cotD
, which contains GerE binding sites that do not overlap with its −35 region.
Structural Characterization of the Lactoferrin Receptor from
Neisseria meningitidis
Authors: Prinz Thorsten , Meyer Markus , Pettersson Annika , Tommassen Jan
Published: December 2019

The meningococcal lactoferrin receptor is composed of the integral outer membrane protein LbpA and the peripheral lipoprotein LbpB. Homooligomeric complexes of LbpA and heterooligomers consisting of LbpA and LbpB were identified. Furthermore, five cell surface-exposed loops of LbpA were identified, which partially confirms a previously proposed topology model.
Growth Phase-Dependent Regulation of Vsr Endonuclease May Contribute to 5-Methylcytosine Mutational Hot Spots in
Escherichia coli
Authors: Macintyre Georgina , Pitsikas Photini , Cupples Claire G.
Published: December 2019

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of
Escherichia coli
is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.
Role of TolR N-Terminal, Central, and C-Terminal Domains in Dimerization and Interaction with TolA and TolQ
Authors: Journet Laure , Rigal Alain , Lazdunski Claude , Bénédetti Hélène
Published: December 2019

The Tol-PAL system of
Escherichia coli
is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.
Characterization of an Atypical Superoxide Dismutase from
Sinorhizobium meliloti
Authors: Santos Renata , Bocquet Stephane , Puppo Alain , Touati Danièle
Published: December 2019

Sinorhizobium meliloti
Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a
sod
fragment amplified by PCR from degenerate primers as a probe. The
sodA
gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical
M
r
of 22,430 and pI of 5.8.
S. meliloti
SOD complemented a deficient
E. coli
mutant, restoring aerobic growth of a
sodA sodB recA
strain, when the gene was expressed from the synthetic
tac
promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H
2
O
2
. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of
S. meliloti
. Activity was restored by dialysis against either MnCl
2
or Fe(NH
4
)
2
(SO
4
)
2
, demonstrating the cambialistic nature of the
S. meliloti
SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H
2
O
2
. Sequence comparison with 70 FeSODs and MnSODs indicates that
S. meliloti
SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H
2
O
2
of this unusual Fe-type SOD.
The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement of
Escherichia coli
Inner Membrane Proteins
Authors: Newitt John A. , Ulbrandt Nancy D. , Bernstein Harris D.
Published: December 2019

The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the
Escherichia coli
inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.
Cloning, Sequencing, and Characterization of the
cgmB
Gene of
Sinorhizobium meliloti
Involved in Cyclic β-Glucan Biosynthesis
Authors: Wang Ping , Ingram-Smith Cheryl , Hadley Jill A. , Miller Karen J.
Published: December 2019

Periplasmic cyclic β-glucans of
Rhizobium
species provide important functions during plant infection and hypo-osmotic adaptation. In
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an
S. meliloti
Tn
5
insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn
5
flanking sequences (amplified by inverse PCR) as a probe, an
S. meliloti
genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb
Hin
dIII-
Eco
RI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform
Rhizobium leguminosarum
bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an
S. meliloti
phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of
S. meliloti
is designated the
cgm
(cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.
OxyR and SoxRS Regulation of
fur
Authors: Zheng Ming , Doan Bernard , Schneider Thomas D. , Storz Gisela
Published: December 2019

The cytotoxic effects of reactive oxygen species are largely mediated by iron. Hydrogen peroxide reacts with iron to form the extremely reactive and damaging hydroxyl radical via the Fenton reaction. Superoxide anion accelerates this reaction because the dismutation of superoxide leads to increased levels of hydrogen peroxide and because superoxide elevates the intracellular concentration of iron by attacking iron-sulfur proteins. We found that regulators of the
Escherichia coli
responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake. A transcript encoding Fur was induced by hydrogen peroxide in a wild-type strain but not in a Δ
oxyR
strain, and DNase I footprinting assays showed that OxyR binds to the
fur
promoter. In cells treated with the superoxide-generating compound paraquat, we observed the induction of a longer transcript encompassing both
fur
and its immediate upstream gene
fldA
, which encodes a flavodoxin. This polycistronic mRNA is induced by paraquat in a wild-type strain but not in a Δ
soxRS
strain, and SoxS was shown to bind to the
fldA
promoter. These results demonstrate that iron metabolism is coordinately regulated with the oxidative stress defenses.
Influence of a Putative ECF Sigma Factor on Expression of the Major Outer Membrane Protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens
Authors: Brinkman Fiona S. L. , Schoofs Geert , Hancock Robert E. W. , De Mot René
Published: December 2019

The gene encoding OprF, a major outer membrane protein inPseudomonasspecies (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene,sigX, located immediately upstream ofoprFin bothPseudomonas aeruginosaPAO1 andPseudomonas fluorescensOE 28.3 and show that disruption of this gene significantly reduces OprF expression. InP. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronicoprFcombined with a polar effect due to termination of a transcript containingsigXandoprF. Comparison ofsigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription ofoprFoccurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of theP. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes inP. aeruginosa. Disruption of thesigXgene inP. fluorescensalso had an effect on the logarithmic-phase growth rate in rich medium. A conservedsigXgene was also identified in aPseudomonas syringaeisolate and sixP. aeruginosaclinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.
Toxicity of Copper, Cobalt, and Nickel Salts Is Dependent on Histidine Metabolism in the YeastSaccharomyces cerevisiae
Authors: Pearce David A. , Sherman Fred
Published: December 2019

The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeastSaccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of thehis1-his7mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His+strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His−strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H+-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.
Hemoglobinase Activity of the Lysine Gingipain Protease (Kgp) of Porphyromonas gingivalis W83
Authors: Lewis Janina P. , Dawson Janet A. , Hannis James C. , Muddiman David , Macrina Francis L.
Published: December 2019

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases ofP. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation ofP. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the α chain and 10 Lys residues in the β chain. In contrast, there are only three Arg residues in each of the α and β chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalisis a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.
The pur7 Gene from the Puromycin Biosynthetic pur Cluster of Streptomyces alboniger Encodes a Nudix Hydrolase
Authors: Espinosa J. C. , Tercero J. A. , Rubio M. A. , Jiménez A.
Published: December 2019

Pur7 is the product of a gene from the puromycin biosyntheticpurcluster ofStreptomyces alboniger. It was expressed inEscherichia colias a recombinant protein fused to a His tag and then was highly purified through a Ni2+column. It showed a 3′-amino-3′-dATP pyrophosphohydrolase (nudix) activity which produced 3′-amino-3′-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg2+. Among a large variety of other nucleotides tested, only 3′-amino-3′-dTTP was a Pur7 substrate, although at lower reaction rates than 3′-amino-3′-dATP. These findings suggest that Pur7 has a high specificity for the 3′ amino group at the ribofuranoside moiety of these two substrates. TheKmandVmaxvalues for these dATP and dTTP derivatives were 120 μM and 17 μM/min and 3.45 mM and 12.5 μM/min, respectively. Since it is well known that 3′-amino-3′-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3′-amino-3′-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.
InvF Is Required for Expression of Genes Encoding Proteins Secreted by the SPI1 Type III Secretion Apparatus inSalmonella typhimurium
Authors: Heran Darwin K. , Miller Virginia L.
Published: December 2019

The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of theinv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion ininvF was constructed and tested for the expression of Φ(sigD-lacZYA),sipC::Tn5lacZY, and a plasmid-encoded Φ(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion ofSalmonella typhimurium into cultured eucaryotic cells.sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of thesigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.
Regulation of the lic Operon ofBacillus subtilis and Characterization of Potential Phosphorylation Sites of the LicR Regulator Protein by Site-Directed Mutagenesis
Authors: Tobisch Steffen , Stülke Jörg , Hecker Michael
Published: December 2019

Thelicoperon ofBacillus subtilisis required for the transport and degradation of oligomeric β-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. Thelicoperon is transcribed from a ςA-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of thelicBCAHpromoter. Expression of thelicoperon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EIILicin the absence of oligomeric β-glucosides. This was shown by the analysis oflicRmutants affected in potential phosphorylation sites. Moreover, thelicoperon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.
Cloning, Characterization, and Expression of a Novel Gene Encoding a Reversible 4-Hydroxybenzoate Decarboxylase from Clostridium hydroxybenzoicum
Authors: Huang Jianbin , He Hongqi , Wiegel Juergen
Published: December 2019

A novel gene, designated ohb1, which encodes the oxygen-sensitive and biotin-, ATP-, thiamin-, pyridoxal phosphate-, and metal-ion-independent, reversible 4-hydroxybenzoate decarboxylase (4-HOB-DC) from the obligate anaerobe Clostridium hydroxybenzoicum JW/Z-1T was sequenced (GenBank accession no. AF128880) and expressed. The 1,440-bp open reading frame (ORF) (ohb1) encodes 480 amino acids. Major properties of the heterologous enzyme (Ohb1) expressed in Escherichia coli DH5α were the same as those described for the native 4-HOB-DC (Z. He and J. Wiegel, J. Bacteriol. 178:3539–3543, 1996). The deduced amino acid sequence shows up to 57% identity and up to 74% similarity to hypothetical proteins deduced from ORFs in genomes from bacteria and archaea, suggesting a possible novel gene family.
Purification and Ligand Binding of EmrR, a Regulator of a Multidrug Transporter
Authors: Brooun Alexei , Tomashek John J. , Lewis Kim
Published: December 2019

EmrR, the repressor of theemrRABoperon ofEscherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump—2,4-dinitrophenol, carbonyl cyanidem-chlorophenylhydrazone, and carbonyl cyanidep-(trifluoro-methoxy)phenylhydrazone—with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.
16S rRNA Is Bound to Era of
Streptococcus pneumoniae
Authors: Meier Timothy I. , Peery Robert B. , Jaskunas S. Richard , Zhao Genshi
Published: December 2019

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione
S
-transferase (GST) fusion forms of
Streptococcus pneumoniae
Era in
Escherichia coli
and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of
S. pneumoniae
during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [

k
D

],

K
m

, and
V
max
). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar

k
D

. In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from
S. pneumoniae
was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.
The 17-Gene Ethanolamine (
eut
) Operon of
Salmonella typhimurium
Encodes Five Homologues of Carboxysome Shell Proteins
Authors: Kofoid Eric , Rappleye Chad , Stojiljkovic Igor , Roth John
Published: December 2019

The
eut
operon of
Salmonella typhimurium
encodes proteins involved in the cobalamin-dependent degradation of ethanolamine. Previous genetic analysis revealed six
eut
genes that are needed for aerobic use of ethanolamine; one (
eutR
), encodes a positive regulator which mediates induction of the operon by vitamin B
12
plus ethanolamine. The DNA sequence of the
eut
operon included 17 genes, suggesting a more complex pathway than that revealed genetically. We have correlated an open reading frame in the sequence with each of the previously identified genes. Nonpolar insertion and deletion mutations made with the Tn
10
-derived transposable element T-POP showed that at least 10 of the 11 previously undetected
eut
genes have no Eut phenotype under the conditions tested. Of the dispensable
eut
genes, five encode apparent homologues of proteins that serve (in other organisms) as shell proteins of the carboxysome. This bacterial organelle, found in photosynthetic and sulfur-oxidizing bacteria, may contribute to CO
2
fixation by concentrating CO
2
and excluding oxygen. The presence of these homologues in the
eut
operon of
Salmonella
suggests that CO
2
fixation may be a feature of ethanolamine catabolism in
Salmonella
.
Comparative Genetics of Capsular Polysaccharide Biosynthesis in
Streptococcus pneumoniae
Types Belonging to Serogroup 19
Authors: Morona Judy K. , Morona Renato , Paton James C.
Published: December 2019

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in
Streptococcus pneumoniae
serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19
cps
loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (
cps19aI
and
cps19fI
). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two
cps
loci (
cps19cS
), which is likely to encode a glucosyl transferase. The arrangement of the genes within the
cps19
loci is highly conserved, with 13 genes (
cps19A
to -
H
and
cps19K
to -
O
) common to all four serogroup 19 members. These
cps
genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19
cps
loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the
cps19
gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.
An Engineered Cytochrome
b
6
c
1
Complex with a Split Cytochrome
b
Is Able To Support Photosynthetic Growth of
Rhodobacter capsulatus
Authors: Saribas A. Sami , Mandaci Sevnur , Daldal Fevzi
Published: December 2019

The ubihydroquinone-cytochrome
c
oxidoreductase (or the cytochrome
bc
1
complex) from
Rhodobacter capsulatus
is composed of the Fe-S protein, cytochrome
b
, and cytochrome
c
1
subunits encoded by
petA
(
fbcF
),
petB
(
fbcB
), and
petC
(
fbcC
) genes organized as an operon. In the work reported here,
petB
(
fbcB
) was split genetically into two cistrons,
petB6
and
petBIV
, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome
b
, respectively. These polypeptides resembled the cytochrome
b
6
and su IV subunits of chloroplast cytochrome
b
6
f
complexes, and together with the unmodified subunits of the cytochrome
bc
1
complex, they formed a novel enzyme, named cytochrome
b
6
c
1
complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of
R. capsulatus
. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of
petBIV
, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome
b
6
c
1
complex were similar to those of the cytochrome
bc
1
complex. In particular, it was highly sensitive to inhibitors of the cytochrome
bc
1
complex, including antimycin A, and the redox properties of its
b
- and
c
-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome
b
L
heme was modified in a way reminiscent of that of a cytochrome
b
6
f
complex. Based on the work described here and that with
Rhodobacter sphaeroides
(R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280–16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome
bc
1
complexes and the chloroplast cytochrome
b
6
f
complexes are direct consequences of splitting cytochrome
b
into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome
bc
oxidoreductases.
Comparison of the
d
-Glutamate-Adding Enzymes from Selected Gram-Positive and Gram-Negative Bacteria
Authors: Walsh Ann W. , Falk Paul J. , Thanassi Jane , Discotto Linda , Pucci Michael J. , Ho Hsu-Tso
Published: December 2019

The biochemical properties of the
d
-glutamate-adding enzymes (MurD) from
Escherichia coli
,
Haemophilus influenzae
,
Enterococcus faecalis
, and
Staphylococcus aureus
were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (
murD
) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein–MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain
d
-glutamate-adding activity and have

K
m

and
V
max
values similar to those of the regenerated MurDs, except for the
H. influenzae
enzyme. Substrate inhibition by UDP-
N
-acetylmuramyl-
l
-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 μM for E. coli and
H. influenzae
MurD, respectively. Such substrate inhibition was not observed with the
E. faecalis
and
S. aureus
enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH
4
+
and/or K
+
, but not Na
+
, for optimal activity, while anions such as Cl
−
and SO
4
2−
had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg
2+
for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.
The Frequency of Conjugative Transposition of Tn
916
Is Not Determined by the Frequency of Excision
Authors: Marra Diana , Pethel Beth , Churchward Gordon G. , Scott June R.
Published: December 2019

Excision and formation of a covalently closed circular transposon molecule are required for conjugative transposition of Tn
916
but are not the only factors that limit the frequency of conjugative transposition from one host to another. We found that in gram-positive bacteria, an increase in the frequency of excision and circularization of Tn
916
caused by expression of integrase (Int) and excisionase (Xis) from a xylose-inducible promoter does not lead to an increase in the frequency of conjugative transposition. We also found that the concentration of Int and Xis in the recipient cell does not limit the frequency of conjugative transposition and that increased excision does not result in increased expression of transfer functions required to mobilize a plasmid containing the Tn
916
origin of transfer. We conclude that in gram-positive hosts in which the Tn
916
functions Int and Xis are overexpressed, the frequency of conjugative transposition is limited by the availability of transfer functions.
ZmaR, a Novel and Widespread Antibiotic Resistance Determinant That Acetylates Zwittermicin A
Authors: Stohl Elizabeth A. , Brady Sean F. , Clardy Jon , Handelsman Jo
Published: December 2019

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of
Bacillus
. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of
E. coli
containing
zmaR
inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by
1
H NMR,
13
C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [
14
C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.
Molecular Characterization of the PhoP-PhoQ Two-Component System in
Escherichia coli
K-12: Identification of Extracellular Mg
2+
-Responsive Promoters
Authors: Kato Akinori , Tanabe Hiroyuki , Utsumi Ryutaro
Published: December 2019

We identified Mg
2+
-responsive promoters of the
phoPQ
,
mgtA
, and
mgrB
genes of
Escherichia coli
K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.
A Hyperactive NAD(P)H:Rubredoxin Oxidoreductase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ma Kesen , Adams Michael W. W.
Published: December 2019

NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the hyperthermophilic archaeon
Pyrococcus furiosus
. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of rubredoxin, a small (5.3-kDa) iron-containing redox protein that had previously been purified from this organism. The apparent
V
max
at 80°C is 20,000 μmol/min/mg, which corresponds to a
k
cat
/

K
m

value of 300,000 mM
−1
s
−1
. The apparent

K
m

values measured at 80°C and pH 8.0 for rubredoxin, NADPH, and NADH were 50, 5, and 34 μM, respectively. The enzyme did not reduce
P. furiosus
ferredoxin. NROR is a monomer with a molecular mass of 45 kDa and contains one flavin adenine dinucleotide molecule per mole but lacks metals and inorganic sulfide. The possible physiological role of this hyperactive enzyme is discussed.