bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Comparison of
fliC
Genes and Identification of Glycosylation in the Flagellin of
Pseudomonas aeruginosa
a-Type Strains
Authors: Brimer Cynthia D. , Montie T. C.
Published: December 2019

Pseudomonas aeruginosa
a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several
Pseudomonas aeruginosa
a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.
The Glucose Kinase of
Bacillus subtilis
Authors: Skarlatos Pierre , Dahl Michael K.
Published: December 2019

The open reading frame
yqgR
(now termed
glcK
), which had been sequenced as part of the genome project, encodes a glucose kinase of
Bacillus subtilis
. A 1.1-kb DNA fragment containing
glcK
complemented an
Escherichia coli
strain deficient in glucose kinase activity. Insertional mutagenesis of
glcK
resulted in a complete inactivation of glucose kinase activity in crude protein extracts, indicating that
B. subtilis
contains one major glucose kinase. The
glcK
gene encodes a 321-residue protein with a molecular mass of 33.5 kDa. The glucose kinase was overexpressed as a fusion protein to a six-His affinity tag and purified to homogeneity. The enzyme had

K
m

values for ATP and glucose of 0.77 and 0.24 mM, respectively, and a
V
max
of 93 μmol min
−1
mg
−1
. A
B. subtilis
strain deficient for glucose kinase grew at the same rate on different carbon sources tested, including disaccharides such as maltose, trehalose, and sucrose.
Mutagenesis and Expression of
amo
, Which Codes for Ammonia Monooxygenase in
Nitrosomonas europaea
Authors: Hommes Norman G. , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: December 2019

Nitrosomonas europaea
has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of
amoA
were obtained, and they were found to differ by one nucleotide. To determine if both copies of
amoA
were functional, insertional mutagenesis was performed to inactivate either copy of
amoA
alone. A DNA cassette containing the
lacZ
and
kan
genes inserted into
amoA
was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of
amoA
. Mutations were obtained in both copies of
amoA
. Either copy of
amoA
was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of
amoA
, but not the other, resulted in slower growth. Measurements of ammonia-dependent O
2
consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [
14
C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.
Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(
RS
)-2-(2,4-Dichlorophenoxy)propanoic acid] by
Sphingomonas herbicidovorans
MH
Authors: Zipper Christian , Bunk Monika , Zehnder Alexander J. B. , Kohler Hans-Peter E.
Published: December 2019

Sphingomonas herbicidovorans
MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(
RS
)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (
S
) enantiomer over the (
R
) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(
RS
)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (

K
t

) of 108, 93, and 117 μM and maximal velocities (
V
max
) of 19, 10, and 21 nmol min
−1
mg of protein
−1
for (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D, respectively. Transport of (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor
N
,
N
′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (
R
)-dichlorprop uptake was inhibited by (
R
)-mecoprop but not by (
S
)-mecoprop, (
S
)-dichlorprop, or 2,4-D. On the other hand, the (
S
)-dichlorprop transport was inhibited by (
S
)-mecoprop but not by (
R
)-mecoprop, (
R
)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by
S. herbicidovorans
is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (
R
)-dichlorprop and (
R
)-mecoprop, another for (
S
)-dichlorprop and (
S
)-mecoprop, and a third for 2,4-D.
Identification of a Specific Chaperone for SptP, a Substrate of the Centisome 63 Type III Secretion System of
Salmonella typhimurium
Authors: Fu Yixin , Galán Jorge E.
Published: December 2019

Salmonella typhimurium
uses of a type III protein secretion system encoded at centisome 63 of its chromosome to deliver effector molecules into the host cell. These proteins stimulate host cell responses such as reorganization of the actin cytoskeleton and activation of transcription factors. One of these effector proteins is SptP, a tyrosine phosphatase that causes disruption of the host cell actin cytoskeleton. A characteristic feature of many substrates of type III secretion systems is their association with specific cytoplasmic chaperones which appears to be required for secretion and/or translocation of these proteins into the host cell. We report here the identification of SicP, a 13-kDa acidic polypeptide that is encoded immediately upstream of
sptP
. A loss-of-function mutation in
sicP
resulted in drastically reduced levels of SptP but did not affect
sptP
expression, indicating that SicP exerts its effect posttranscriptionally. Pulse-chase experiments demonstrated that the loss of SicP leads to increased degradation of SptP. In addition, we show that SicP binds to SptP directly and that the binding site is located between residues 15 and 100 of the tyrosine phosphatase. Taken together, these results indicate that SicP acts as a specific chaperone for SptP.
Cloning, Sequencing, and Phenotypic Characterization of the
rpoS
Gene from
Pseudomonas putida
KT2440
Authors: Ramos-González María Isabel , Molin Søren
Published: December 2019

A gene homologous to the
rpoS
gene of
Escherichia coli
was cloned from a
Pseudomonas putida
KT2440 gene bank by complementation of the
rpoS
-deficient strain
E. coli
ZK918. The
rpoS
gene of
P. putida
complemented the acid sensitivity and catalase deficiency of the
rpoS
mutant of
E. coli
and stimulated expression of the RpoS-controlled promoter,
bolAp
1
. The gene was sequenced and found to be highly similar to the
rpoS
genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of the
nlpD
gene was found upstream to the
rpoS
gene. A transcriptional fusion of the promoter of the
P. putida rpoS
gene to the
luxAB
genes from
Vibrio harveyi
was constructed and used as an inactivated allele of
rpoS
for gene replacement of the wild-type copy in the chromosome of
P. putida
. The resultant
rpoS
mutant of
P. putida
, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with
m
-methylbenzoate was also reduced in the mutant strain
P. putida
C1R1. The RpoS protein of
P. putida
controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.
Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine
Authors: Burke Stephen A. , Lo Sam L. , Krzycki Joseph A.
Published: December 2019

Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in
Methanosarcina barkeri
MS. The gene encoding MMCP (
mtmC
) is located directly upstream of the gene encoding MMAMT (
mtmB
). The gene encoding MT2-A (
mtbA
) was found 1.1 kb upstream of
mtmC
, but no obvious open reading frame was found in the intergenic region between
mtbA
and
mtmC
. A single monocistronic transcript was found for
mtbA
that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried
mtmCB
. The larger transcript also encoded
mtmP
, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of
mtmC
. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of
mtmB
was interrupted by a single in-frame, midframe, UAG codon which was also found in
mtmB
from
M. barkeri
NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of
mtmB
in this methanogen.
Characterization of the Earliest Known
Staphylococcus aureus
Plasmid Encoding a Multidrug Efflux System
Authors: Paulsen Ian T. , Brown Melissa H. , Skurray Ronald A.
Published: December 2019

The staphylococcal
qacB
-encoding multidrug resistance plasmid pSK156, isolated from a clinical strain dating from 1951, was characterized. Comparison of the regions flanking
qacB
with other
qacA
- and
qacB
-encoding plasmids provided insights into the evolution and dissemination of these multidrug efflux genes and led to the detection of the earliest known copy of the insertion sequence IS
257
.
Characterization of the Maleylacetate Reductase MacA of
Rhodococcus opacus
1CP and Evidence for the Presence of an Isofunctional Enzyme
Authors: Seibert Volker , Kourbatova Elena M. , Golovleva Ludmila A. , Schlömann Michael
Published: December 2019

Maleylacetate reductases (EC
1.3.1.32
) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene
macA
, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain
Rhodococcus opacus
1CP. Sequencing of
macA
showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned
Rhodococcus
maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of
macA
, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with
macA
encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the
R. opacus
maleylacetate reductase gene
macA
clearly is not part of a specialized chlorocatechol gene cluster.
The
apeE
Gene of
Salmonella typhimurium
Encodes an Outer Membrane Esterase Not Present in
Escherichia coli
Authors: Carinato Maria E. , Collin-Osdoby Patricia , Yang Xioming , Knox Tina M. , Conlin Christopher A. , Miller Charles G.
Published: December 2019

Salmonella typhimurium apeR
mutations lead to overproduction of an outer membrane-associated
N
-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the
apeE
gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the
S. typhimurium apeE
gene as well as some properties of the esterase that it encodes. The predicted product of
apeE
is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium
Photorhabdus luminescens
. The
Salmonella
esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C
6
to C
16
fatty acid
p
-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing
Salmonella
spp. from related organisms makes use of the ability of
Salmonella
to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the
apeE
gene product is the enzyme in
Salmonella
uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that
Escherichia coli
K-12 does not contain a close analog of
apeE
, and it appears that the
apeE
gene is contained in a region of DNA present in
Salmonella
but not in
E. coli
.
Dual Promoters Are Responsible for Transcription Initiation of the
fla/che
Operon in
Bacillus subtilis
Authors: Estacio W. , Anna-Arriola S. Santa , Adedipe M. , Márquez-Magaña L. M.
Published: December 2019

The
fla/che
region contains more than 30 genes required for flagellar synthesis and chemotaxis in
Bacillus subtilis
, including the gene for the flagellum-specific ς
D
factor,
sigD
. Sequence and primer extension data demonstrate that a P
A
promoter immediately upstream of
flgB
, henceforth referred to as the
fla/che
P
A
, and the P
D-3
promoter are active in vivo. Transcription from the P
D-3
element is dependent on ς
D
activity and is regulated by the flagellum-specific negative regulator, FlgM. In a strain containing a deletion of
fla/che
P
A
(P
A
Δ), ς
D
protein was not detected, demonstrating that the
fla/che
P
A
is necessary for wild-type expression of the
sigD
gene. Thus,
sigD
is part of the >26-kb
fla/che
operon. Consistent with a lack of detectable ς
D
protein, the P
A
Δ strain grows as long filaments and does not express a ς
D
-dependent
hag
::
lacZ
reporter construct. These phenotypes are indicative of a lack of
sigD
expression or complete inhibition of ς
D
activity by FlgM. However, ς
D
activity is found in a double mutant containing the P
A
Δ and a null mutation in
flgM
. The double mutant no longer grows as long filaments, and expression of
hag
::
lacZ
is partially restored. These data demonstrate that a low level of ς
D
activity does exist in the P
A
Δ mutant but can be detected only in the presence of a null mutation in
flgM
. Therefore, normal expression of
sigD
may also involve another promoter(s) within the
fla/che
operon.
Spo0A Mutants of
Bacillus subtilis
with Sigma Factor-Specific Defects in Transcription Activation
Authors: Hatt Janet K. , Youngman Philip
Published: December 2019

The transcription factor Spo0A of
Bacillus subtilis
has the unique ability to activate transcription from promoters that require different forms of RNA polymerase holoenzyme. One class of Spo0A-activated promoter, which includes
spoIIEp
, is recognized by RNA polymerase associated with the primary sigma factor, sigma A (ς
A
); the second, which includes
spoIIAp
, is recognized by RNA polymerase associated with an early-sporulation sigma factor, sigma H (ς
H
). Evidence suggests that Spo0A probably interacts directly with RNA polymerase to activate transcription from these promoters. To identify residues of Spo0A that may be involved in transcriptional activation, we used PCR mutagenesis of the entire
spo0A
gene and designed a screen using two distinguishable reporter fusions,
spoIIE-gus
and
spoIIA-lacZ
. Here we report the identification and characterization of five mutants of Spo0A that are specifically defective in activation of ς
A
-dependent promoters while maintaining activation of ς
H
-dependent promoters. These five mutants identify a 14-amino-acid segment of Spo0A, from residue 227 to residue 240, that is required for transcriptional activation of ς
A
-dependent promoters. This region may define a surface or domain of Spo0A that makes direct contacts with ς
A
-associated holoenzyme.
A Minisatellite Sequence within the Propeptide Region of the Vacuolar Carboxypeptidase Y Gene of
Schizosaccharomyces pombe
Authors: Ingavale Susham S. , Kaur Rupinder , Aggarwal Parul , Bachhawat Anand K.
Published: December 2019

We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast
Schizosaccharomyces pombe
. The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene. The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of
S. pombe
(
S. pombe
var.
pombe
and
S. pombe
var.
malidevorans
). The minisatellite sequence, however, appeared to be species specific and was absent in other members of the
Schizosaccharomyces
genus. This report constitutes the first experimental demonstration of the presence of such sequences in yeasts.
General Stress Transcription Factor ς
B
and Sporulation Transcription Factor ς
H
Each Contribute to Survival of
Bacillus subtilis
under Extreme Growth Conditions
Authors: Gaidenko Tatiana A. , Price Chester W.
Published: December 2019

The general stress response of the bacterium
Bacillus subtilis
is controlled by the ς
B
transcription factor. Here we show that loss of ς
B
reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol. We further show that loss of the developmental transcription factor ς
H
also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.
Biochemical Properties and Substrate Specificities of a Recombinantly Produced
Azotobacter vinelandii
Alginate Lyase
Authors: Ertesvåg Helga , Erlien Frode , Skjåk-Bræk Gudmund , Rehm Bernd H. A. , Valla Svein
Published: December 2019

Alginate is a polysaccharide composed of β-
d
-mannuronic acid (M) and α-
l
-guluronic acid (G). An
Azotobacter vinelandii
alginate lyase gene,
algL
, was cloned, sequenced, and expressed in
Escherichia coli
. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from
Pseudomonas aeruginosa
. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the

K
m

was found to be 4.6 × 10
−4
M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.
Expression of the
fixR-nifA
Operon in
Bradyrhizobium japonicum
Depends on a New Response Regulator, RegR
Authors: Bauer Evelyne , Kaspar Thomas , Fischer Hans-Martin , Hennecke Hauke
Published: December 2019

Many nitrogen fixation-associated genes in the soybean symbiont
Bradyrhizobium japonicum
are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the
fixR-nifA
operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension. This induction depends on a −24/−12-type promoter (
fixRp
1
) that is recognized by the ς
54
RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (
fixRp
2
) that overlaps with
fixRp
1
and depends on an upstream DNA region (UAS) located around position −68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760–1765, 1995). A protein binding to the UAS was previously postulated to act as an activator. This protein has now been purified, and the corresponding gene (
regR
) has been cloned. On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems. We identified upstream of the
regR
gene an additional gene (
regS
) encoding a putative sensor kinase. A
regR
mutant was constructed in which neither a specific UAS-binding activity nor
fixRp
2
-dependent transcript formation and
fixR′-′lacZ
expression was detected in aerobically grown cells. Anaerobic
fixR′-′lacZ
expression was also decreased in
regR
mutants to about 10% of the level observed in the wild type. Similarly,
regR
mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by
nifA
mutants, the morphology of those nodules was normal, displaying no signs of necrosis. While
regR
mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of
regS
mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains. The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling
nif
and
fix
gene expression in
B. japonicum
.
Activation Control of
pur
Gene Expression in
Lactococcus lactis
: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of
purC
and
purD
Promoter Regions
Authors: Kilstrup Mogens , Jessing Stine G. , Wichmand-Jørgensen Stephanie B. , Madsen Mette , Nilsson Dan
Published: December 2019

A comparison of the
purC
and
purD
upstream regions from
Lactococcus lactis
revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς
70
promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in
purC
is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the
purC
and
purD
promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea that
purC
and
purD
transcription is regulated by a transcriptional activator binding to the PurBox sequence.
Structural and Biochemical Analysis of the Sheath of
Phormidium uncinatum
Authors: Hoiczyk Egbert
Published: December 2019

The sheath of the filamentous, gliding cyanobacterium
Phormidium uncinatum
was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in
P. uncinatum
directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699–708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In
P. uncinatum
the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.
The Flagellar Switch Genes
fliM
and
fliN
of
Rhodobacter sphaeroides
Are Contained in a Large Flagellar Gene Cluster
Authors: García Norma , Campos Andrés , Osorio Aurora , Poggio Sebastian , González-Pedrajo Bertha , Camarena Laura , Dreyfus Georges
Published: December 2019

In this work, the genes that encode the FliM and FliN proteins of
Rhodobacter sphaeroides
were characterized. These genes are part of a large flagellar gene cluster in which six additional open reading frames encoding products homologous to FliL, FliO, FliP, FliQ, FliR, and FlhB proteins from other bacteria were identified. The inactivation of the
fliM
gene gave a nonflagellate phenotype (Fla
−
), suggesting that FliM is required for flagellar assembly. Complementation analysis of this
fliM
mutant indicated that
fliM
and
fliN
transcription starts beyond the 5′ end of
fliK
and terminates after
fliN
.
Construction and Physiological Analysis of a
Xanthomonas
Mutant To Examine the Role of the
oxyR
Gene in Oxidant-Induced Protection against Peroxide Killing
Authors: Mongkolsuk Skorn , Sukchawalit Rojana , Loprasert Suvit , Praituan Wipa , Upaichit Apichat
Published: December 2019

We constructed and characterized a
Xanthomonas campestris
pv. phaseoli
oxyR
mutant. The mutant was hypersensitive to H
2
O
2
and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and
ahpC
genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the
oxyR
mutant. These results show that
X. campestris
pv. phaseoli
oxyR
is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.
Cloning, Characterization, and Transcriptional Analysis of a Gene Encoding an α-Crystallin-Related, Small Heat Shock Protein from the Thermophilic Cyanobacterium
Synechococcus vulcanus
Authors: Roy Sanjit Kumer , Nakamoto Hitoshi
Published: December 2019

hspA
, a gene encoding a 16-kDa heat-induced protein from the thermophilic cyanobacterium
Synechococcus vulcanus
, has been cloned and sequenced. The deduced amino acid sequence of the gene product showed significant homology to sequences of the family of α-crystallin-related, small heat shock proteins. A monocistronic mRNA of
hspA
increased transiently in response to heat shock. The heat shock induction occurred at a vegetative promoter but without the CIRCE (controlling inverted repeat of chaperone expression) element.
A Silent ABC Transporter Isolated from
Streptomyces rochei
F20 Induces Multidrug Resistance
Authors: Fernández-Moreno Miguel A. , Carbó Lázaro , Cuesta Trinidad , Vallín Carlos , Malpartida Francisco
Published: December 2019

In the search for heterologous activators for actinorhodin production in
Streptomyces lividans
, 3.4 kb of DNA from
Streptomyces rochei
F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the
actII-ORF4
gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3′ end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3′ terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named
msr
, is a new bacterial multidrug ABC transporter.
Molecular Cloning and Functional Expression of a Protein-Serine/Threonine Phosphatase from the Hyperthermophilic Archaeon
Pyrodictium abyssi
TAG11
Authors: Mai Bianca , Frey Gerhard , Swanson Ronald V. , Mathur Eric J. , Stetter K. O.
Published: December 2019

An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon
Pyrodictium abyssi
TAG11 and named Py-PP1. Py-PP1 was expressed in
Escherichia coli
, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo.
Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of
Saccharomyces cerevisiae
Authors: Small W. Curtis , McAlister-Henn Lee
Published: December 2019

The reoxidation of NADH generated in reactions within the mitochondrial matrix of
Saccharomyces cerevisiae
is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated
NDH1
results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated
NDH2
has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of
NDH1
and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.
Insertion Mutation of the Form I
cbbL
Gene Encoding Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in
Thiobacillus neapolitanus
Results in Expression of Form II RuBisCO, Loss of Carboxysomes, and an Increased CO
2
Requirement for Growth
Authors: Baker Stefanie H. , Jin Songmu , Aldrich Henry C. , Howard Gary T. , Shively Jessup M.
Published: December 2019

It has been previously established that
Thiobacillus neapolitanus
fixes CO
2
by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme,
cbbL
and
cbbS
, are part of a putative carboxysome operon. In the present study,
cbbL
and
cbbS
were cloned and sequenced. Analysis of RNA showed that
cbbL
and
cbbS
are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into
cbbL
resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO
2
, but requires a CO
2
supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO
2
by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.
Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from
Sphingomonas
sp. Strain RW5
Authors: Werwath Jörn , Arfmann Hans-Adolf , Pieper Dietmar H. , Timmis Kenneth N. , Wittich Rolf-Michael
Published: December 2019

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC
1.13.11.4
),
gtdA
, from
Sphingomonas
sp. strain RW5 was cloned and sequenced. The
gtdA
gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the
gtdA
gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (
phdI
) of the phenanthrene degradation in
Nocardioides
sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in
E. coli
. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (

K
m

) and specificity constants (
K
cat
/
K
m
) of the enzyme were determined to be 15 μM and 511 s
−1
M
−1
× 10
4
for gentisate and 754 μM and 20 s
−1
M
−1
× 10
4
for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of
gtdA
. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.
Localization and Function of Early Cell Division Proteins in Filamentous
Escherichia coli
Cells Lacking Phosphatidylethanolamine
Authors: Mileykovskaya Eugenia , Sun Qin , Margolin William , Dowhan William
Published: December 2019

Escherichia coli
cells that contain the
pss-93
null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in
pss-93
filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.
The
Escherichia coli
Starvation Gene
cstC
Is Involved in Amino Acid Catabolism
Authors: Fraley C. D. , Kim J. H. , McCann M. P. , Matin A.
Published: December 2019

Escherichia coli
strains mutant in the starvation gene
cstC
grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources. They are also compromised in starvation survival, where amino acid catabolism is important. The
cstC
gene encodes a 406-amino-acid protein that closely resembles the
E. coli
ArgD protein, which is involved in arginine biosynthesis. We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway. The
cstC
upstream region contains several regulatory consensus sequences. Both ς
S
and ς
54
promoters are probably involved in
cstC
transcription and appear to compete with each other, presumably to match
cstC
expression to the cellular amino acid catabolic needs.
Cloning and DNA Sequencing of the Genes Encoding
Clostridium josui
Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome
Authors: Kakiuchi Motohide , Isui Ayako , Suzuki Katsuhisa , Fujino Tsuchiyoshi , Fujino Emi , Kimura Tetsuya , Karita Shuichi , Sakka Kazuo , Ohmiya Kunio
Published: December 2019

The
Clostridium josui cipA
and
celD
genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of
C. josui
CipA is similar to those of the scaffolding proteins reported so far, such as
Clostridium thermocellum
CipA,
Clostridium cellulovorans
CbpA, and
Clostridium cellulolyticum
CipC, but
C. josui
CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the
C. josui
cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.
Mechanism for Biotransformation of Nonylphenol Polyethoxylates to Xenoestrogens in
Pseudomonas putida
Authors: John Dominic M. , White Graham F.
Published: December 2019

A strain of
Pseudomonas putida
isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEO

x

, where
x
is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEO
av6
, NPEO
av9
, and NPEO
av20
(mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO
2
). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEO

x

. Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEO

x

mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEO

x

-degrading activity was inducible by substrate, and cell extracts of NPEO
av9
-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO
8
), producing sequentially, under either aerobic or anaerobic conditions, AEO
7
, AEO
6
, AEO
5
, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEO
av9
or AEO
8
under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEO

x

and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEO

x
−1

, which then undergoes further cycles of the same reaction until
x
= 2.
Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in
Lactobacillus johnsonii
100-100
Authors: Elkins Christopher A. , Savage Dwayne C.
Published: December 2019

Cytosolic extracts of
Lactobacillus johnsonii
100-100 (previously reported as
Lactobacillus
sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in
Escherichia coli
. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily.
E. coli
DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [
14
C]taurocholic acid that were increased as much as threefold over control levels. [
14
C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.
Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from
Pseudomonas
sp. Strain HR199 and Molecular Characterization of the Gene
Authors: Achterholt Sandra , Priefert Horst , Steinbüchel Alexander
Published: December 2019

The coniferyl aldehyde dehydrogenase (CALDH) of
Pseudomonas
sp. strain HR199 (DSM7063), which catalyzes the NAD
+
-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α
2
structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The

K
m

values for coniferyl aldehyde and NAD
+
were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH
2
-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (
calB
). The corresponding nucleotide sequence was localized on a 9.4-kbp
Eco
RI fragment (E94), which was subcloned from a
Pseudomonas
sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD
+
-dependent aldehyde dehydrogenases from different sources.