bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Duplication and Multiplicity of Collagenases in
Clostridium histolyticum
Authors: Matsushita Osamu , Jung Chang-Min , Katayama Seiichi , Minami Junzaburo , Takahashi Yukie , Okabe Akinobu
Published: December 2019

Clostridium histolyticum
collagenase contains a number of different active components. Previously we have shown that
colH
encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (
colG
). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that
colG
and
colH
encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome.
C. histolyticum colG
is more similar to
C. perfringens colA
than to
colH
in terms of domain structure. Both
colG
and
colA
have a homologous gene,
mscL
, at their 3′ ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to
C. histolyticum
and
C. perfringens
and that further divergence of the parent gene produced
colG
and
colA
.
The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from
Klebsiella aerogenes
and
Escherichia coli
Authors: Muse Wilson B. , Bender Robert A.
Published: December 2019

The nitrogen assimilation control protein (NAC) from
Klebsiella aerogenes
or
Escherichia coli
(NAC
K
or NAC
E
, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (
hut
) operon and to repress transcription of the glutamate dehydrogenase (
gdh
) operon in
K. aerogenes
. Truncated NAC polypeptides, generated by the introduction of stop codons within the
nac
open reading frame, were tested for the ability to activate
hut
and repress
gdh
in vivo. Most of the NAC
K
and NAC
E
fragments with 100 or more amino acids (wild-type NAC
K
and NAC
E
both have 305 amino acids) were functional in activating
hut
and repressing
gdh
expression in vivo. Full-length NAC
K
and NAC
E
were isolated as chimeric proteins with the maltose-binding protein (MBP). NAC
K
and NAC
E
released from such chimeras were able to activate
hut
transcription in a purified system in vitro, as were NAC
K
129 and NAC
E
100 (a NAC
K
fragment of 129 amino acids and a NAC
E
fragment of 100 amino acids) released from comparable chimeras. A set of NAC
E
and NAC
K
fragments carrying nickel-binding histidine tags (his
6
) at their C termini were also generated. All such constructs derived from NAC
E
were insoluble, as was NAC
E
itself. Of the his
6
-tagged constructs derived from NAC
K
, NAC
K
100 was inactive, but NAC
K
120 was active. Several NAC fragments were tested for dimerization. NAC
K
120-his
6
and NAC
K
100-his
6
were dimers in solution. MBP-NAC
K
and MBP-NAC
K
129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NAC
E
was readily cleaved by factor Xa, but the resulting NAC
E
was also degraded by the protease. However, MBP-NAC
E
-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.
EmrE, a Small
Escherichia coli
Multidrug Transporter, Protects
Saccharomyces cerevisiae
from Toxins by Sequestration in the Vacuole
Authors: Yelin Rodrigo , Rotem Dvir , Schuldiner Shimon
Published: December 2019

In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from
Escherichia coli
, in the yeast
Saccharomyces cerevisiae
. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP
+
), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up
14
C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu
2+
ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in
14
C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu
2+
and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu
2+
, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.
Physiological Characterization of
Pseudomonas aeruginosa
during Exotoxin A Synthesis: Glutamate, Iron Limitation, and Aconitase Activity
Authors: Somerville Greg , Mikoryak Carole Ann , Reitzer Larry
Published: December 2019

Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from
Pseudomonas aeruginosa
. We tested the possibility that glutamate affects growth during iron restriction. We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate. We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation. Glutamate had no effect on
toxA
(ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells. In contrast, citrate and isocitrate diminished
toxA
transcription. Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle. Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control. There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis. We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility. Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo. In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase. We speculate that one or more of the aconitases in
P. aeruginosa
may contribute to the control of virulence factor synthesis.
Genetic and Transcriptional Analyses of the
Vibrio cholerae
Mannose-Sensitive Hemagglutinin Type 4 Pilus Gene Locus
Authors: Marsh Jane W. , Taylor Ronald K.
Published: December 2019

The mannose-sensitive hemagglutinin (MSHA) of the
Vibrio cholerae
O1 El Tor biotype is a member of the family of type 4 pili. Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer. The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the
V. cholerae
chromosome. Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified. Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes. This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci. The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB of
Escherichia coli
. In
E. coli
, the
yhdA
and
mreB
genes are adjacent to each other on the chromosome. The finding that the MSHA locus lies between these two
E. coli
homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.
Identification of a
Streptococcus pneumoniae
Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System
Authors: Novak Rodger , Cauwels Anje , Charpentier Emmanuelle , Tuomanen Elaine
Published: December 2019

The
Escherichia coli
Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in
E. coli
including a gene encoding a PhoU protein. Similar to
E. coli
, mutagenesis of the ATP-binding cassette gene,
pstB
, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant
phoU
. By using a
lacZ
reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of
E. coli.
Analysis of
rpoS
mRNA in
Salmonella dublin
: Identification of Multiple Transcripts with Growth-Phase-Dependent Variation in Transcript Stability
Authors: Paesold G. , Krause M.
Published: December 2019

In
Salmonella dublin
,
rpoS
encodes an alternative sigma factor of the RNA polymerase that activates a variety of stationary-phase-induced genes, including some virulence-associated genes. In this work, we studied the regulation and transcriptional organization of
rpoS
during growth. We found two transcripts, 2.3 and 1.6 kb in length, that represent the complete
rpoS
sequence. The 2.3-kb transcript is a polycistronic message that also includes the upstream
nlpD
gene. It is driven by a weak promoter with increasing activity when cells enter early stationary growth. The 1.6-kb message includes 566 bp upstream of the
rpoS
start codon. It is transcribed from a strong ς
70
RNA polymerase-dependent promoter which is independent of growth. The decay of this transcript decreases substantially in early stationary growth, resulting in a significant net increase in
rpoS
mRNA levels. These levels are approximately 10-fold higher than the levels of the 2.3-kb mRNA, indicating that the 1.6-kb message is mainly responsible for RpoS upregulation. In addition to the 2.3- and 1.6-kb transcripts, two smaller 1.0- and 0.4-kb RNA species are produced from the
nlpD-rpoS
locus. They do not allow translation of full-length RpoS; hence their significance for
rpoS
regulation remains unclear. We conclude that of four transcripts arising from the
nlpD-rpoS
locus, only one plays a significant role in
rpoS
expression in
S. dublin
. Its upregulation when cells enter stationary growth is due primarily to an increase in transcript stability.
Functional Determinants of the
Escherichia coli fis
Promoter: Roles of −35, −10, and Transcription Initiation Regions in the Response to Stringent Control and Growth Phase-Dependent Regulation
Authors: Walker Kimberly A. , Atkins Carey L. , Osuna Robert
Published: December 2019

Escherichia coli
Fis is a small DNA binding and bending protein that has been implicated in a variety of biological processes. A minimal promoter sequence consisting of 43 bp is sufficient to generate its characteristic growth phase-dependent expression pattern and is also subject to negative regulation by stringent control. However, information about the precise identification of nucleotides contributing to basal promoter activity and its regulation has been scant. In this work, 72 independent mutations were generated in the
fis
promoter (
fis
P) region from −108 to +78 using both random and site-directed PCR mutagenesis. β-Galactosidase activities from mutant promoters fused to the (
trp-lac
)
W200
fusion on a plasmid were used to conclusively identify the sequences TTTCAT and TAATAT as the −35 and −10 regions, respectively, which are optimally separated by 17 bp. We found that four consecutive substitutions within the GC-rich sequence just upstream of +1 and mutations in the −35 region, but not in the −10 region, significantly reduced the response to stringent control. Analysis of the effects of mutations on growth phase-dependent regulation showed that replacing the predominant transcription initiation nucleotide +1C with a preferred nucleotide (A or G) profoundly altered expression such that high levels of
fis
P mRNA were detected during late logarithmic and early stationary phases. A less dramatic effect was seen with improvements in the −10 and −35 consensus sequences. These results suggest that the acute growth phase-dependent regulation pattern observed with this promoter requires an inefficient transcription initiation process that is achieved with promoter sequences deviating from the −10 and −35 consensus sequences and, more importantly, a dependence upon the availability of the least favored transcription initiation nucleotide, CTP.
Molecular Cloning, Sequencing, Purification, and Characterization of
Pseudomonas aeruginosa
Ribosome Recycling Factor
Authors: Ohnishi Makoto , Janosi Laszlo , Shuda Masahiro , Matsumoto Hideki , Hayashi Tetsuya , Terawaki Yoshiro , Kaji Akira
Published: December 2019

Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (
frr
) of
Pseudomonas aeruginosa
PAO1 was functionally cloned by using a temperature-sensitive
frr
mutant of
Escherichia coli
and sequenced. The
P. aeruginosa frr
was mapped at 30 to 32 min of the
P. aeruginosa
chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of
E. coli
RRF. In an assay including
E. coli
polysome and elongation factor G, purified recombinant RRF of
P. aeruginosa
released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (
rpsB
), elongation factor Ts (
tsf
), and UMP kinase (
pyrH
) are located upstream of
frr
. The arrangement of the genes,
rpsB-tsf-pyrH-frr
, resembles those reported for
E. coli
and
Bacillus subtilis
. Even in the cyanobacterium genome, the arrangement
pyrH-frr
is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.
Auxins Upregulate Expression of the Indole-3-Pyruvate Decarboxylase Gene in
Azospirillum brasilense
Authors: Vande Broek Ann , Lambrecht Mark , Eggermont Kristel , Vanderleyden Jos
Published: December 2019

Transcription of the
Azospirillum brasilense ipdC
gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by
ipdC-gusA
expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, and
p
-chlorophenoxyacetic acid were also found to upregulate
ipdC
expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA-
l
-phenylalanine, indicating structural specificity is required for
ipdC
induction. This is the first report describing the induction of a bacterial gene by auxin.
Bacteriophage T4 gp2 Interferes with Cell Viability and with Bacteriophage Lambda Red Recombination
Authors: Appasani Krishnarao , Thaler David S. , Goldberg Edward B.
Published: December 2019

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.
The Virulence Plasmid of
Salmonella typhimurium
Is Self-Transmissible
Authors: Ahmer Brian M. M. , Tran Mimi , Heffron Fred
Published: December 2019

Most isolates of
Salmonella enterica
serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10
−4
transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.
3-Hydroxylaminophenol Mutase from
Ralstonia eutropha
JMP134 Catalyzes a Bamberger Rearrangement
Authors: Schenzle Andreas , Lenke Hiltrud , Spain Jim C. , Knackmuss Hans-Joachim
Published: December 2019

3-Hydroxylaminophenol mutase from
Ralstonia eutropha
JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH
2
-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.
Mechanism of Citrate Metabolism in
Lactococcus lactis
: Resistance against Lactate Toxicity at Low pH
Authors: Magni Christian , de Mendoza Diego , Konings Wil N. , Lolkema Juke S.
Published: December 2019

Measurement of the flux through the citrate fermentation pathway in resting cells of
Lactococcus lactis
CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in
L. lactis
is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of
L. lactis
in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.
Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in
Salmonella typhimurium
Authors: Nambu Takayuki , Minamino Tohru , Macnab Robert M. , Kutsukake Kazuhiro
Published: December 2019

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in
Salmonella typhimurium
. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from
S. typhimurium
has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed
flgJ
mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.
Direct Selection for Mutators in
Escherichia coli
Authors: Miller Jeffrey H. , Suthar Anjali , Tai Jennifer , Yeung Annie , Truong Cindy , Stewart Jean Lee
Published: December 2019

We have constructed strains that allow a direct selection for mutators of
Escherichia coli
on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (
mutHLS
and
uvrD
). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10
−5
in the population. Also, we can measure a mutator cascade, in which one type of mutator (
mutT
) generates a second mutator (
mutHLS
) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.
Complete Sequence of a 184-Kilobase Catabolic Plasmid from
Sphingomonas aromaticivorans
F199
Authors: Romine Margaret F. , Stillwell Lisa C. , Wong Kwong-Kwok , Thurston Sarah J. , Sisk Ellen C. , Sensen Christoph , Gaasterland Terry , Fredrickson Jim K. , Saffer Jeffrey D.
Published: December 2019

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from
Sphingomonas aromaticivorans
F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene,
m
-xylene, and
p
-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another
Sphingomonas
sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.
The P

alkBFGHJKL

Promoter Is under Carbon Catabolite Repression Control in
Pseudomonas oleovorans
but Not in
Escherichia coli alk
+
Recombinants
Authors: Staijen Ivo E. , Marcionelli Rosanna , Witholt Bernard
Published: December 2019

The
alk
genes are located on the OCT plasmid of
Pseudomonas oleovorans
and encode an inducible pathway for the utilization of
n
-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in
P. oleovorans
and
Escherichia coli alk
+
recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The
alk
genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both
E. coli
and pseudomonads.
Pseudomonas putida
GPo12 is a
P. oleovorans
derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant
E. coli
HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the
alkB
gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in
Pseudomonas
and in
E. coli
strains. These results also indicate that P

alkBFGHJKL

, the P

alk

promoter, might be useful in attaining high expression levels of heterologous genes in
E. coli
grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.
Characterization of the Exopolygalacturonate Lyase PelX ofErwinia chrysanthemi3937
Authors: Shevchik Vladimir E. , Kester Harry C. M. , Benen Jacques A. E. , Visser Jaap , Robert-Baudouy Janine , Hugouvieux-Cotte-Pattat Nicole
Published: December 2019

Erwinia chrysanthemi3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-α-d-galacturonosidase, PehX, have been previously identified in otherE. chrysanthemistrains. Using a genomic bank of a 3937 mutant with the majorpelgenes deleted, we cloned a pectinase gene identified aspelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of anotherE. chrysanthemistrain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL ofE. chrysanthemibut has a N-terminal extension of 324 residues. The transcription ofpelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation ofpelXexpression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved inpelXexpression. ThepelXmutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers orSaintpaulia ionanthaplants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites −2 to +2. PelX and PehX were shown to be localized in the periplasm ofE. chrysanthemi3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.
Identification of Mechanosensitive Ion Channels in the Cytoplasmic Membrane of
Corynebacterium glutamicum
Authors: Ruffert Susanne , Berrier Catherine , Krämer Reinhard , Ghazi Alexandre
Published: December 2019

Patch-clamp experiments performed on membrane fragments of
Corynebacterium glutamicum
fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.
Identification and Characterization of a DeoR-Specific Operator Sequence Essential for Induction of
dra-nupC-pdp
Operon Expression in
Bacillus subtilis
Authors: Zeng Xianmin , Saxild Hans H.
Published: December 2019

The
deoR
gene located just upstream the
dra-nupC-pdp
operon of
Bacillus subtilis
encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional
lacZ
fusions. It was shown that all of the
cis
-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of
dra
. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position −60 to position −43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3′ half of the palindrome and was located between the −10 and −35 regions of the
dra
promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the
dra-nupC-pdp
expression.
Tyrosine Aminotransferase Catalyzes the Final Step of Methionine Recycling in
Klebsiella pneumoniae
Authors: Heilbronn Jacqueline , Wilson Judith , Berger Bradley J.
Published: December 2019

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified from
Klebsiella pneumoniae
and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the
Escherichia coli
and
Salmonella typhimurium
tyrosine aminotransferases (
tyrB
gene products). The structural gene for the tyrosine aminotransferase was cloned from
K. pneumoniae
and expressed in
E. coli
. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from
E. coli
,
S. typhimurium
,
Paracoccus denitrificans
, and
Rhizobium meliloti
, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the
K. pneumoniae
enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.
CspA, CspB, and CspG, Major Cold Shock Proteins of
Escherichia coli
, Are Induced at Low Temperature under Conditions That Completely Block Protein Synthesis
Authors: Etchegaray Jean-Pierre , Inouye Masayori
Published: December 2019

CspA, CspB, and CspG, the major cold shock proteins of
Escherichia coli
, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37°C in the presence of kanamycin (100 μg/ml) or chloramphenicol (200 μg/ml). After 10 min of incubation with the antibiotics at 37°C, cells were cold shocked at 15°C and labeled with [
35
S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of
cspA
and
cspB
mRNAs for the first hour at 15°C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.
Aspartate 205 in the Catalytic Domain of Naphthalene Dioxygenase Is Essential for Activity
Authors: Parales Rebecca E. , Parales Juanito V. , Gibson David T.
Published: December 2019

The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonassp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571–586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one α subunit and mononuclear iron in the adjacent α subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene tocis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAPand reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.
Cloning of
mnuA
, a Membrane Nuclease Gene of
Mycoplasma pulmonis
, and Analysis of Its Expression in
Escherichia coli
Authors: Jarvill-Taylor Karalee J. , VanDyk Christina , Minion F. Chris
Published: December 2019

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from
Mycoplasma pulmonis
. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in
Escherichia coli
. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated
mnuA
(for “membrane nuclease”). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an
M. pulmonis
membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with
E. coli
confirmed that
mnuA
coded for a nuclease of unknown specificity. Hybridization studies showed that
mnuA
sequences are found in few
Mycoplasma
species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus
Mycoplasma
.
Characterization of the Divergent
sacBK
and
sacAR
Operons, Involved in Sucrose Utilization by
Lactococcus lactis
Authors: Luesink Evert J. , Marugg Joey D. , Kuipers Oscar P. , de Vos Willem M.
Published: December 2019

The divergently transcribed
sacBK
and
sacAR
operons, which are involved in the utilization of sucrose by
Lactococcus lactis
NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing
sacB
and
sacK
, a second of 3.4 kb containing
sacA
and
sacR
, and a third of 1.8 kb containing only
sacR
. The inactivation of the
sacR
gene by replacement recombination resulted in the constitutive transcription of the
sacBK
and
sacAR
operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription.
LB-AUT7
, a Novel Symbiosis-Regulated Gene from an Ectomycorrhizal Fungus,
Laccaria bicolor
, Is Functionally Related to Vesicular Transport and Autophagocytosis
Authors: Kim Sung-Jae , Bernreuther Daniela , Thumm Michael , Podila Gopi K.
Published: December 2019

We have identified
LB-AUT7
, a gene differentially expressed 6 h after ectomycorrhizal interaction between
Laccaria bicolor
and
Pinus resinosa
. LB-Aut7p can functionally complement its
Saccharomyces cerevisiae
homolog, which is involved in the attachment of autophagosomes to microtubules. Our findings suggest the induction of an autophagocytosis-like vesicular transport process during ectomycorrhizal interaction.
Escherichia coli
Outer Membrane Protein TolC Is Involved in Production of the Peptide Antibiotic Microcin J25
Authors: Delgado Mónica A. , Solbiati José O. , Chiuchiolo María J. , Farías Ricardo N. , Salomón Raúl A.
Published: December 2019

A Tn
5
insertion in
tolC
eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The
tolC
mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.
Twelve
pil
Genes Are Required for Biogenesis of the R64 Thin Pilus
Authors: Yoshida Tetsu , Kim Su-Ryang , Komano Teruya
Published: December 2019

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes,
pilI
to -
V
, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594–3603, 1997). In this study, we introduced frameshift mutations into each of the 14
pil
genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes,
pilK
to -
V
, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes,
traBC
, were previously shown to be required for the expression of the
pil
genes. In addition, the
rci
gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes,
traBC
,
pilK
through
pilV
, and
rci
, are essential for R64 thin pilus formation and function.
HmbR, a Hemoglobin-Binding Outer Membrane Protein of
Neisseria meningitidis
, Undergoes Phase Variation
Authors: Richardson Anthony R. , Stojiljkovic Igor
Published: December 2019

Neisseria meningitidis
uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the
hmbR
expression pattern in strains in which the
hpuB
gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some
hpuB
mutants is suggestive of phase variation of
hmbR
. The length of the poly(G) tract starting at position +1164 of
hmbR
absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring the
hmbR
reading frame. Inactivation of
hmbR
in these strains resulted in a loss of Hb utilization, demonstrating that the change in the
hmbR
gene was responsible for the phenotypic switch. The switching rates from
hmbR
phase off to phase on were ∼5 × 10
−4
in four serogroup C strains, 2 × 10
−2
in the serogroup A isolate, and 7 × 10
−6
in the serogroup B isolate.
Stabilization of the Relaxosome and Stimulation of Conjugal Transfer Are Genetically Distinct Functions of the R1162 Protein MobB
Authors: Perwez Tariq , Meyer Richard J.
Published: December 2019

MobB is a small protein encoded by the broad-host-range plasmid R1162 and required for efficient mobilization of its DNA during conjugation. The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the origin of transfer (
oriT
). We have generated in-frame
mobB
deletions that specifically inactivate the stabilizing effect of MobB while still allowing a high rate of transfer. Thus, MobB has two genetically distinct functions in transfer. The effect of another deletion, extending into
mobA
, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain. The
mobB
mutations that specifically affected stability also resulted in poor growth of cells, due to increased transcription from the promoters adjacent to
oriT
. The effects of the mutations could be suppressed not only by full-length MobB provided in
trans
, as expected, but also by additional copies of
oriT
, cloned in pBR322. In addition, in the presence of MobA both the full-length and truncated forms of MobB stimulated recombination between
oriT
-containing plasmids. We propose a model in which MobB regulates expression of plasmid genes by altering the stability of the relaxosome, in a manner that involves the coupling of plasmid molecules.