bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Regulation of Genes Encoding Arabinan-Degrading Enzymes in
Bacillus subtilis
Authors: Raposo Maria Paiva , Inácio José Manuel , Mota Luís Jaime , de Sá-Nogueira Isabel
Published: February 2004

Bacillus subtilis
produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The
abfA
gene comprised in the metabolic operon
araABDLMNPQ
-
abfA
and the
xsa
gene located 23 kb downstream most probably encode α-
l
-arabinofuranosidases (EC 3.2.1.55). Here, we show that the
abnA
gene, positioned immediately upstream from the metabolic operon, encodes an endo-α-1,5-arabinanase (EC 3.2.1.99). Furthermore, by in vivo RNA studies, we inferred that
abnA
and
xsa
are monocistronic and are transcribed from σ
A
-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of
xsa
and
abnA
. The results indicate that transcriptional repression of the
abfA
and
xsa
genes is achieved by a tightly controlled mechanism but that the regulation of
abnA
is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.
Chitinase B of “
Microbulbifer degradans
” 2-40 Contains Two Catalytic Domains with Different Chitinolytic Activities
Authors: Howard Michael B. , Ekborg Nathan A. , Taylor Larry E. , Weiner Ronald M. , Hutcheson Steven W.
Published: February 2004

Chitinase B of “
Microbulbifer degradans
” 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18
N
) releases methylumbelliferone from 4′-methylumbelliferyl-
N
,
N
′-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18
C
) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18
C
releases methylumbelliferone from 4′-methylumbelliferyl-
N
,
N
′,
N
"-triacetylchitotriose 2.7-fold faster than GH18
N
and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37°C and from pH 7.2 to 8.0 and was not affected by Mg
2+
, Mn
2+
, Ca
2+
, K
+
, EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni
2+
, Sr
2+
, and Cu
2+
, while Hg
2+
completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.
In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination
Authors: Torres Martha , Balada Joan-Miquel , Zellars Malcolm , Squires Craig , Squires Catherine L.
Published: February 2004

Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rho-dependent terminator. A comparison of terminator read-through in a wild-type
Escherichia coli
strain and that in a
nusB
::IS
10
mutant strain determined the requirement for NusB. In the absence of NusB, antiterminator-dependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusG-depleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.
Identification and Mapping of Self-Assembling Protein Domains Encoded by theEscherichia coliK-12 Genome by Use of λ Repressor Fusions
Authors: Mariño-Ramírez Leonardo , Minor Jonathan L. , Reading Nicola , Hu James C.
Published: February 2004

Self-assembling proteins and protein fragments encoded by theEscherichia coligenome were identified fromE. coliK-12 strain MG1655. Libraries of random DNA fragments cloned into a series of λ repressor fusion vectors were subjected to selection for immunity to infection by phage λ. Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232E. coligenes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online athttp://oligomers.tamu.edu/.
Functionally Critical Elements of CooA-Related CO Sensors
Authors: Youn Hwan , Kerby Robert L. , Conrad Mary , Roberts Gary P.
Published: February 2004

CooA is a heme-containing transcriptional activator that enables
Rhodospirillum rubrum
to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in
Escherichia coli
, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from
Azotobacter vinelandii
and
Carboxydothermus hydrogenoformans
corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of
R. rubrum
. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.
Dimerization of the RamC Morphogenetic Protein of
Streptomyces coelicolor
Authors: Hudson Michael E. , Nodwell Justin R.
Published: February 2004

RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium
Streptomyces coelicolor
. This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanism of action. In this study we found that the presence of multiple copies of a defective allele of
ramC
inhibits morphogenesis in
S. coelicolor
, consistent with either titration of a target or formation of inactive RamC multimers. We identified a domain in RamC that is C terminal to the putative kinase domain and forms a dimer with a

K
d

of ∼0.1 μM. These data suggest that RamC acts as a dimer in vivo.
The
Pseudomonas putida
Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds
Authors: Morales Gracia , Linares Juan Francisco , Beloso Ana , Albar Juan Pablo , Martínez José Luis , Rojo Fernando
Published: February 2004

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in
Pseudomonas putida
and
Pseudomonas aeruginosa
when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic
P. putida
strains containing either a wild-type or an inactivated
crc
allele was compared. The results showed that Crc is involved in the catabolic repression of the
hpd
and
hmgA
genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in
P. putida
. It was found that genes required to assimilate benzoate through the catechol pathway (
benA
and
catBCA
) and 4-OH-benzoate through the protocatechuate pathway (
pobA
and
pcaHG
) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in
P. putida
.
In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of
Streptomyces noursei
ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis
Authors: Sekurova Olga N. , Brautaset Trygve , Sletta Håvard , Borgos Sven E. F. , Jakobsen Øyvind M. , Ellingsen Trond E. , Strøm Arne R. , Valla Svein , Zotchev Sergey B.
Published: February 2004

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in
Streptomyces noursei
ATCC 11455. Gene inactivation and complementation experiments revealed that
nysRI
,
nysRII
,
nysRIII
, and
nysRIV
are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the
xylE
reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the
S. noursei
wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that
nysR
genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.
Expression of Cholera Toxin under Non-AKI Conditions in
Vibrio cholerae
El Tor Induced by Increasing the Exposed Surface of Cultures
Authors: Sánchez Joaquín , Medina Gerardo , Buhse Thomas , Holmgren Jan , Soberón-Chavez Gloria
Published: February 2004

The regulatory systems controlling expression of the
ctxAB
genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic
Vibrio cholerae
have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.
In Silico and Transcriptional Analysis of Carbohydrate Uptake Systems of
Streptomyces coelicolor
A3(2)
Authors: Bertram Ralph , Schlicht Maximilian , Mahr Kerstin , Nothaft Harald , Saier Milton H. , Titgemeyer Fritz
Published: February 2004

Streptomyces coelicolor
is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of
S. coelicolor
A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, α-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and β-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and
N
-acetylglucosamine, the genome of
S. coelicolor
encodes at least 53 potential carbohydrate uptake systems.
Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in
Xanthomonas oryzae
pv. oryzae
Authors: Furutani Ayako , Tsuge Seiji , Ohnishi Kouhei , Hikichi Yasufumi , Oku Takashi , Tsuno Kazunori , Inoue Yasuhiro , Ochiai Hirokazu , Kaku Hisatoshi , Kubo Yasuyuki
Published: February 2004

Xanthomonas oryzae
pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient
hrp
-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of
X. oryzae
pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the
hrp
regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by
hrp
genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that
cysP2
has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a
cysP2
::
gus
fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the
hrp
regulatory gene
hrpXo
is also involved in the expression of not only
hrp
genes and type III secretory proteins but also some type II secretory proteins.
Studies of the Interaction of
Escherichia coli
YjeQ with the Ribosome In Vitro
Authors: Daigle Denis M. , Brown Eric D.
Published: February 2004

Escherichia coli
YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of
E. coli
and
Bacillus subtilis
. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h
−1
) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry
41:
11109-11117, 2002). In the work reported here, YjeQ from
E. coli
was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5′-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.
Transcriptional Organization and Regulation of the
l
-Idonic Acid Pathway (GntII System) in
Escherichia coli
Authors: Bausch Christoph , Ramsey Matthew , Conway Tyrrell
Published: February 2004

The genetic organization of the
idn
genes that encode the pathway for
l
-idonate catabolism was characterized. The monocistronic
idnK
gene is transcribed divergently from the
idnDOTR
genes, which were shown to form an operon. The 215-bp regulatory region between the
idnK
and
idnD
genes contains promoters in opposite orientation with transcription start sites that mapped to positions −26 and −29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of
idnD
, and an UP element upstream of
idnK
. The genes of the
l
-idonate pathway were shown to be under catabolite repression control. Analysis of
idnD
- and
idnK
-
lacZ
fusions in a nonpolar
idnD
mutant that is unable to interconvert
l
-idonate and 5-ketogluconate indicated that either compound could induce the pathway. The
l
-idonate pathway was first characterized as a subsidiary pathway for
d
-gluconate catabolism (GntII), which is induced by
d
-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the
idnK
and
idnD
operons are induced by
d
-gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from
d
-gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in
l
-idonate catabolism; the GntII system can be induced by
d
-gluconate under conditions that block the GntI system.
Relevance of Peptide Uptake Systems to the Physiology and Virulence of
Streptococcus agalactiae
Authors: Samen Ulrike , Gottschalk Birgit , Eikmanns Bernhard J. , Reinscheid Dieter J.
Published: February 2004

Streptococcus agalactiae
is a major cause of invasive infections in human newborns. To satisfy its growth requirements,
S. agalactiae
takes up 9 of the 20 proteinogenic amino acids from the environment. Defined
S. agalactiae
mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by
S. agalactiae
was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes
oppA1
-
F
and
dppA
-
E
and a monocistronic organization of
dpsA
in
S. agalactiae
. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons
dppA
-
E
and
oppA1
-
F
in
S. agalactiae
. Growth of
S. agalactiae
in human amniotic fluid was shown to require an intact
dpsA
gene, indicating an important role of DpsA during the infection of the amniotic cavity by
S. agalactiae
. Deletion of the
oppB
gene reduced the adherence of
S. agalactiae
to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the
fbsA
gene, which encodes a fibrinogen-binding protein in
S. agalactiae
. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in
S. agalactiae
.
A Dominant-NegativefurMutation inBradyrhizobium japonicum
Authors: Benson Heather P. , LeVier Kristin , Guerinot Mary Lou
Published: February 2004

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiontBradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified afurmutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of thefurgene repress iron uptake systems and starve for iron, whereasfurmutants fail to repress iron uptake systems and survive. TheB. japonicum furmutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of thefurgene cannot complement thefurmutant. Expression of thefurmutant allele in wild-type cells leads to afurphenotype. Unlike aB. japonicum fur-null mutant, the strain carrying the dominant-negativefurmutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.
VirB1 Orthologs fromBrucella suisand pKM101 Complement Defects of the Lytic Transglycosylase Required for Efficient Type IV Secretion fromAgrobacterium tumefaciens
Authors: Höppner Christoph , Liu Zhenying , Domke Natalie , Binns Andrew N. , Baron Christian
Published: February 2004

Type IV secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells. The translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions. However, orthologs to proteins of the prototypical type IV system, VirB ofAgrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved. We here report a heterologous complementation in the case ofA. tumefaciens virB1defects with its orthologs fromBrucella suis(VirB1s) and the IncN plasmid pKM101 (TraL). In contrast, expression of the genes encoding the VirB1 orthologs from the IncF plasmid (open reading frame 169) and from theHelicobacter pylori cagpathogenicity island (HP0523) did not complement VirB1 functions. The complementation of VirB1 activity was assessed by T-pilus formation, by tumor formation on wounded plants, by IncQ plasmid transfer, and by IncQ plasmid recipient assay. Replacement of the key active-site Glu residue by Ala abolished the complementation by VirB1 fromB. suisand by TraL, demonstrating that heterologous complementation requires an intact lytic transglycosylase active site. In contrast, the VirB1 active-site mutant fromA. tumefaciensretained considerable residual activity in various activity assays, implying that this protein exerts additional effects during the type IV secretion process.
Role of Histone-Like Protein H-NS in Multidrug Resistance of
Escherichia coli
Authors: Nishino Kunihiko , Yamaguchi Akihito
Published: February 2004

The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in
hns
result in various phenotypes, but the role of H-NS in the drug resistance of
Escherichia coli
has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the
hns
gene from the Δ
acrAB
mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the
hns
mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the
hns
deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene
tolC
. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of
acrEF
,
mdtEF
, and
emrKY
was observed in the Δ
hns
strain by quantitative real-time reverse transcription-PCR analysis. The Δ
hns
-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the
acrEF
gene greatly suppressed the level of Δ
hns
-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the
mdtEF
and
acrEF
genes completely suppressed Δ
hns
-mediated multidrug resistance, indicating that Δ
hns
-mediated multidrug resistance is due to derepression of the
acrEF
and
mdtEF
drug exporter genes.
TeiR, a LuxR-Type Transcription Factor Required for Testosterone Degradation inComamonas testosteroni
Authors: Pruneda-Paz José Luis , Linares Mauricio , Cabrera Julio E. , Genti-Raimondi Susana
Published: February 2004

We have identified a new steroid-inducible gene (designatedteiR[testosterone-inducible regulator]) inComamonas testosteronithat is required for testosterone degradation. Nucleotide sequence analysis ofteiRpredicts a 391-amino-acid protein which shows homology between residues 327 and 380 (C-terminal domain) to the LuxR helix-turn-helix DNA binding domain and between residues 192 and 227 to the PAS sensor domain. This domain distribution resembles that described for TraR, a specific transcriptional regulator involved in quorum sensing inAgrobacterium tumefaciens. Analysis of the gene expression indicated thatteiRis tightly controlled at the transcriptional level by the presence of testosterone in the culture medium. AteiR-disrupted mutant strain was completely unable to use testosterone as the sole carbon and energy source. In addition, the expression of several steroid-inducible genes was abolished in this mutant. Northern blot assays revealed thatteiRis required for full expression ofsip48-β-HSD gene mRNA (encoding a steroid-inducible protein of 48 kDa and 3β-17β-hydroxysteroid dehydrogenase) and also of other steroid degradation genes, including those encoding 3α-hydroxysteroid dehydrogenase, Δ5-3-ketoisomerase, 3-oxo-steroid Δ1-dehydrogenase, and 3-oxo-steroid Δ4-(5α)-dehydrogenase enzymes. Moreover, whenteiRwas provided to theteiR-disrupted strain intrans, the transcription level of these genes was restored. These results indicate that TeiR positively regulates the transcription of genes involved in the initial enzymatic steps of steroid degradation inC. testosteroni.
Development of Surface Adhesion inCaulobacter crescentus
Authors: Bodenmiller Diane , Toh Evelyn , Brun Yves V.
Published: February 2004

Caulobacter crescentushas a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage,C. crescentusis often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment ofC. crescentusto abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion inC. crescentusis a complex developmental process. We found that the attachment ofC. crescentusto surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.
Transcriptional Profiling ofCaulobacter crescentusduring Growth on Complex and Minimal Media
Authors: Hottes Alison K. , Meewan Maliwan , Yang Desiree , Arana Naomi , Romero Pedro , McAdams Harley H. , Stephens Craig
Published: February 2004

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacteriumCaulobacter crescentusduring growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential inC. crescentusfor growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth ofC. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.
Assembly of an Oxalate Decarboxylase Produced under σ
K
Control into the
Bacillus subtilis
Spore Coat
Authors: Costa Teresa , Steil Leif , Martins Lígia O. , Völker Uwe , Henriques Adriano O.
Published: February 2004

Over 30 polypeptides are synthesized at various times during sporulation in
Bacillus subtilis
, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the
B. subtilis oxdD
gene was previously shown to have oxalate decarboxylase activity when it was produced in
Escherichia coli
and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of
oxdD
is induced during sporulation as a monocistronic unit under the control of σ
K
and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a
cotE
mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of
oxdD
resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.
Cardiolipin Domains in
Bacillus subtilis
Marburg Membranes
Authors: Kawai Fumitaka , Shoda Momoko , Harashima Rie , Sadaie Yoshito , Hara Hiroshi , Matsumoto Kouji
Published: February 2004

Recently, use of the cardiolipin (CL)-specific fluorescent dye 10-
N
-nonyl-acridine orange (NAO) revealed CL-rich domains in the
Escherichia coli
membrane (E. Mileykovskaya and W. Dowhan, J. Bacteriol.
182:
1172-1175, 2000). Staining of
Bacillus subtilis
cells with NAO showed that there were green fluorescence domains in the septal regions and at the poles. These fluorescence domains were scarcely detectable in exponentially growing cells of the
clsA
-disrupted mutant lacking detectable CL. In sporulating cells with a wild-type lipid composition, fluorescence domains were observed in the polar septa and on the engulfment and forespore membranes. Both in the
clsA
-disrupted mutant and in a mutant with disruptions in all three of the paralogous genes (
clsA
,
ywjE
, and
ywiE
) for CL synthase, these domains did not vanish but appeared later, after sporulation initiation. A red shift in the fluorescence due to stacking of two dye molecules and the lipid composition suggested that a small amount of CL was present in sporulating cells of the mutants. Mass spectrometry analyses revealed the presence of CL in these mutant cells. At a later stage during sporulation of the mutants the frequency of heat-resistant cells that could form colonies after heat treatment was lower. The frequency of sporulation of these cells at 24 h after sporulation initiation was 30 to 50% of the frequency of the wild type. These results indicate that CL-rich domains are present in the polar septal membrane and in the engulfment and forespore membranes during the sporulation phase even in a
B. subtilis
mutant with disruptions in all three paralogous genes, as well as in the membranes of the medial septa and at the poles during the exponential growth phase of wild-type cells. The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranes are involved in sporulation.
Bordetella
Species Are Distinguished by Patterns of Substantial Gene Loss and Host Adaptation
Authors: Cummings C. A. , Brinig M. M. , Lepp P. W. , van de Pas S. , Relman D. A.
Published: February 2004

Pathogens of the bacterial genus
Bordetella
cause respiratory disease in humans and animals. Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified. To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysis of genome content in 42
Bordetella
strains by hybridization of genomic DNA to a microarray representing the genomes of three
Bordetella
species and by subtractive hybridization. Here we show that
B. pertussis
and
B. parapertussis
are predominantly differentiated from
B. bronchiseptica
by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen, which may be important determinants of host specificity. The species also exhibit sequence diversity at a number of surface protein-encoding loci, including the fimbrial major subunit gene,
fim2
. Gene loss, rather than gene acquisition, accompanied by the proliferation of transposons, has played a fundamental role in the evolution of the pathogenic bordetellae and may represent a conserved evolutionary mechanism among other groups of microbial pathogens.
Residues Required for
Bacillus subtilis
PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3
Authors: Chen Yinghua , Abdel-Fattah Wael R. , Hulett F. Marion
Published: February 2004

Bacillus subtilis
PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP
I206A
, PhoP
R210A
, PhoP
L209A
, and PhoP
H208A
) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP
H205A
and PhoP
V204A
) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP
V202A
and PhoP
D203A
, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP
V190A
, PhoP
W191A
, PhoP
Y193A
, PhoP
F195A
, PhoP
G197A,
PhoP
T199A
, and PhoP
R200A
) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP
W191A
and PhoP
R200A
could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of
Escherichia coli
.
Genomic and Genetic Analysis of
Bordetella
Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes
Authors: Liu Minghsun , Gingery Mari , Doulatov Sergei R. , Liu Yichin , Hodes Asher , Baker Stephen , Davis Paul , Simmonds Mark , Churcher Carol , Mungall Karen , Quail Michael A. , Preston Andrew , Harvill Eric T. , Maskell Duncan J. , Eiserling Frederick A. , Parkhill Julian , Miller Jeff F.
Published: February 2004

Liu et al. recently described a group of related temperate bacteriophages that infect
Bordetella
subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science
295:
2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the
mtd
(major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences,
bbp36
contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and
bpm
encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these
Bordetella
phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes.
Bordetella
bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.
eBURST: Inferring Patterns of Evolutionary Descent among Clusters of Related Bacterial Genotypes from Multilocus Sequence Typing Data
Authors: Feil Edward J. , Li Bao C. , Aanensen David M. , Hanage William P. , Spratt Brian G.
Published: February 2004

The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain
23F
-1 clonal complex of
Streptococcus pneumoniae
, “population snapshots” of the entire
S. pneumoniae
and
Staphylococcus aureus
MLST databases, and the more complicated clonal complexes observed for
Campylobacter jejuni
and
Neisseria meningitidis
.
Evolution of a Pathway to Novel Long-Chain Carotenoids
Authors: Umeno Daisuke , Arnold Frances H.
Published: February 2004

Using methods of laboratory evolution to force the C
30
carotenoid synthase CrtM to function as a C
40
synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C
45
and C
50
carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in
Escherichia coli
comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.
Identification of a 349-Kilodalton Protein (Gli349) Responsible for Cytadherence and Glass Binding during Gliding of
Mycoplasma mobile
Authors: Uenoyama Atsuko , Kusumoto Akiko , Miyata Makoto
Published: February 2004

Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of
Mycoplasma mobile
were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 ± 200. The antibody inhibited both the hemadsorption and glass binding of
M. mobile
. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.
The Switch I and II Regions of MinD Are Required for Binding and Activating MinC
Authors: Zhou Huaijin , Lutkenhaus Joe
Published: February 2004

MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations inminDand carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.