bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Early Production of CCL20, or Macrophage Inflammatory Protein 3α, by Human Mast Cells in Response toPseudomonas aeruginosa
Authors: Lin Tong-Jun , Maher Lauren H. , Gomi Kaede , McCurdy Jeffrey D. , Garduno Rafael , Marshall Jean S.
Published: December 2002

Mast cells are important as sentinel cells in host defense against bacterial infection. Much of their effectiveness depends upon recruiting other immune cells; however, little is known about the mechanisms of this response. CCL20, also known as macrophage inflammatory protein-3α (MIP-3α), Exodus, and LARC, is a chemokine known to be a potent chemoattractant for immature dendritic cells and T cells. In this study, we examined the human mast cell production of both CCL20 and granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine for innate immune responses in the lung, in response toPseudomonas aeruginosa. Reverse transcription-PCR and Western blot analysis demonstrated that the human mast cells (HMC-1) express CCL20 mRNA and are able to produce a significant amount (32.4 ng/ml) of CCL20 protein following stimulation by calcium ionophore and phorbol myristate acetate. Importantly,P. aeruginosapotently stimulated CCL20 production in human cord blood-derived mast cells (CBMC), with production peaking at 6 h after stimulation. This time course of expression was distinct from that of GM-CSF, which peaked after 24 to 48 h. Significant CCL20 production did not occur following immunoglobulin E-mediated activation of CBMC under conditions which induced a substantial GM-CSF response. Interestingly, the CCL20 response of mast cells toP. aeruginosawas relatively resistant to inhibition by the corticosteroid dexamethasone, interleukin-10, or cyclosporine, while GM-CSF production was potently inhibited. However,P. aeruginosa-induced CCL20 production was blocked by the protein kinase C (PKC) inhibitor Ro 31-8220 and a PKC pseudosubstrate. These results support a role for human mast cells in the initiation of immune responses toP. aeruginosainfection.
The
Salmonella enterica
Serovar Typhimurium Translocated Effectors SseJ and SifB Are Targeted to the
Salmonella
-Containing Vacuole
Authors: Freeman Jeremy A. , Ohl Michael E. , Miller Samuel I.
Published: December 2002

The
Salmonella enterica
serovar Typhimurium type III secretion system (TTSS) encoded in
Salmonella
pathogenicity island 2 (SPI-2) promotes replication within host cells and systemic infection of mice. The SPI-2 TTSS is expressed following
Salmonella
internalization into host cells and translocates effectors across the membrane of the
Salmonella
-containing vacuole (SCV). Two effectors with similar amino-terminal domains, SseJ and SifB, localize to the SCV membrane in infected HEp-2 cells and subsequently traffic away from the SCV along
Salmonella-
induced-filaments (Sifs). Following infection of RAW cells, SseJ and SifB localize to the SCV as well as LAMP-1-positive, vesicular-appearing structures distant from the SCV. Trafficking of SseJ and SifB away from the SCV requires the SPI-2 effector SifA. Deletion of
sseJ
, but not
sifB
, leads to attenuation of
Salmonella
replication in mice following intraperitoneal inoculation. The contribution of SseJ to in vivo replication is identical in wild-type and
sifA
deletion backgrounds, suggesting that SseJ trafficking away from the SCV along Sifs is unnecessary for its virulence function.
Divergent Patterns of Colonization and Immune Response Elicited from Two IntestinalLactobacillusStrains That Display Similar Properties In Vitro
Authors: Ibnou-Zekri Nabila , Blum Stephanie , Schiffrin Eduardo J. , von der Weid Thierry
Published: December 2002

Lactobacilli derived from the endogenous flora of normal donors are being increasingly used as probiotics in functional foods and as vaccine carriers. However, a variety of studies done with distinct strains of lactobacilli has suggested heterogeneous and strain-specific effects. To dissect this heterogeneity at the immunological level, we selected two strains of lactobacilli that displayed similar properties in vitro and studied their impact on mucosal and systemic B-cell responses in monoxenic mice. Germfree mice were colonized withLactobacillus johnsonii(NCC 533) orLactobacillus paracasei(NCC 2461). Bacterial loads were monitored for 30 days in intestinal tissues, and mucosal and systemic B-cell responses were measured. Although bothLactobacillusstrains displayed similar growth, survival, and adherence properties in vitro, they colonized the intestinal lumen and translocated into mucosal lymphoid organs at different densities.L. johnsoniicolonized the intestine very efficiently at high levels, whereas the number ofL. paracaseidecreased rapidly and it colonized at low levels. We determined whether this difference in colonization correlated with an induction of different types of immune responses. We observed that colonization with either strain induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that both strains were able to activate mucosal B-cell responses. However, clear differences in patterns of immunoglobulins were observed between the two strains in the mucosa and in the periphery. Therefore, despite similar in vitro probiotic properties, distinctLactobacillusstrains may colonize the gut differently and generate divergent immune responses.
Differential Cytokine Pattern in the Spleens and Livers of BALB/c Mice Infected withPenicillium marneffei: Protective Role of Gamma Interferon
Authors: Sisto Francesca , Miluzio Annarita , Leopardi Orazio , Mirra Maurizio , Boelaert Johan R. , Taramelli Donatella
Published: December 2002

Penicillium marneffeiis an intracellular opportunistic fungus causing invasive mycosis in AIDS patients. T cells and macrophages are important for protection in vivo. However, the role of T-cell cytokines in the immune response againstP. marneffeiis still unknown. We studied by semiquantitative reverse transcription-PCR and biological assays the patterns of expression of Th1 and Th2 cytokines in the organs of wild-type (wt) and gamma interferon (IFN-γ) knockout (GKO) mice infected intravenously withP. marneffeiconidia. At 3 × 105conidia/mouse, a self-limiting infection developed in wt BALB/c mice, whereas all GKO mice died at day 18 postinoculation. Splenic and hepatic granulomas were present in wt mice, whereas disorganized masses of macrophages and yeast cells were detected in GKO mice. The infection resolved faster in the spleens than in the livers of wt mice and was associated with the local expression of type 1 cytokines (high levels of interleukin-12 [IL-12] and IFN-γ) but not type 2 cytokines (low levels of IL-4 and IL-10). Conversely, both type 1 and type 2 cytokines were detected in the livers of wt animals. Disregulation of the cytokine profile was seen in the spleens but not in the livers of GKO mice. The inducible nitric oxide synthase mRNA level was low and the TNF-α level was high in both spleens and livers of GKO mice compared to wt mice. These data suggest that the polarization of a protective type 1 immune response againstP. marneffeiis regulated at the level of individual organs and that the absence of IFN-γ is crucial for the activation of fungicidal macrophages and the development of granulomas.
Diversity at the Locus Associated with Transcription of a Variable Surface Antigen ofPneumocystis cariniias an Index of Population Structure and Dynamics in Infected Rats
Authors: Keely Scott P. , Cushion Melanie T. , Stringer James R.
Published: December 2002

Pneumocystis cariniiexpresses a surface glycoprotein called MSG. Different isoforms of MSG are encoded by a gene family spread over at least 15 telomeric sites. Only one locus, called UCS, supports the production of MSG mRNA. Previous studies showed thatP. cariniipopulations from individual rats exhibited high degrees of diversity with respect to the MSG genes attached to the UCS locus. This diversity could have been generated primarily in the rats studied. Alternatively, the rats may have been infected byP. cariniiorganisms that were already different at the UCS locus. To investigate this issue, we examined the UCS locus inP. cariniifrom rats that had been exposed to few of the microbes at a specified time, which produced a bottleneck in the microbial population. Some of the rats with bottlenecks producedP. cariniipopulations in which a single MSG sequence resided at the UCS locus in 80 to 90% of the organisms, showing thatP. cariniican proliferate within a rat without generating the very high levels of UCS diversity previously seen. From the degree of diversity observed in the bottlenecked populations, the maximum rate of switching appeared to be 0.01 event per generation. These data also suggest that the infectious dose is as low as one organism, that rats that share a cage readily infect each other, and that the doubling time ofP. cariniiin vivo is ∼3 days. In addition, we found that inoculation with 107P. cariniiorganisms from a population highly heterogeneous at the UCS locus reproduced this heterogeneity. By contrast, shifts in population structure occurred in rats given 104P. cariniiorganisms, suggesting that a small fraction of these proliferated.
Role of Avian Pathogenic
Escherichia coli
Virulence Factors in Bacterial Interaction with Chicken Heterophils and Macrophages
Authors: Mellata Melha , Dho-Moulin Maryvonne , Dozois Charles M. , Curtiss Roy , Lehoux Brigitte , Fairbrother John M.
Published: December 2002

Avian pathogenic
Escherichia coli
(APEC) cause extraintestinal disease in avian species via respiratory tract infection. Virulence factors associated with APEC include type 1 and P fimbriae, curli, aerobactin, lipopolysaccharide (LPS), K1 capsular antigen, temperature-sensitive hemagglutinin (Tsh), and an uncharacterized pathogen-specific chromosomal region (the 0-min region). The role of these virulence factors in bacterial interaction with phagocytes was investigated by using mutants of three APEC strains, each belonging to one of the most predominant serogroups O1, O2, and O78. Bacterial cell interaction with avian phagocytes was tested with primary cultures of chicken heterophils and macrophages. The presence of type 1 fimbriae and, in contrast, the absence of P fimbriae, K1 capsule, O78 antigen, and the 0-min region promoted bacterial association with chicken heterophils and macrophages. The presence of type 1 and P fimbriae, O78 antigen, and the 0-min region seemed to protect bacteria against the bactericidal effect of phagocytes, especially heterophils. The tested virulence factors seemed to have a limited role in intracellular survival for up to 48 h in macrophages. Generally, opsonized and nonopsonized bacteria were eliminated to the same extent, but in some cases, unopsonized bacteria were eliminated to a greater extent than opsonized bacteria. These results confirm the important role of type 1 fimbriae in promotion of initial phagocytosis, but nevertheless indicate a role for type 1 fimbriae in the protection of bacteria from subsequent killing, at least in heterophils. The results also indicate a role for K1 capsule, O78 antigen, P fimbriae, and the 0-min region in initial avoidance of phagocytosis, but demonstrate an additional role for O78 antigen, P fimbriae, and the 0-min region in subsequent protection against the bactericidal effects of phagocytes after bacterial association has occurred.
The
Vibrio
Pathogenicity Island-Encoded Mop Protein Modulates the Pathogenesis and Reactogenicity of Epidemic
Vibrio cholerae
Authors: Zhang Dalin , Xu Zheng , Sun Weiyun , Karaolis David K. R.
Published: December 2002

Epidemic
Vibrio cholerae
possess the VPI (
Vibrio
pathogenicity island) essential virulence gene cluster. The VPI is 41.2 kb in size and encodes 29 potential proteins, several of which have no known function. We show that the VPI-encoded Orf4 is a predicted 34-kDa periplasmic protein containing a zinc metalloprotease motif.
V. cholerae
seventh-pandemic (El Tor) strain N16961 carrying an
orf4
mutation showed no obvious difference relative to its parent in the production of cholera toxin and the toxin-coregulated pilus, motility, azocasein digestion, and colonization of infant mice. However, analysis of rabbit ileal loops revealed that the N16961
orf4
mutant is hypervirulent, causing increased serosal hemorrhage and reactogenicity compared to its parent. Histology revealed a widening of submucosa, with an increase in inflammatory cells, diffuse lymphatic vessel dilatation, edema, endothelial cell hypertrophy of blood vessels, blunting of villi, and lacteal dilatation with lymphocytes and polymorphonuclear leukocytes. The mutant could be complemented in vivo with an
orf4
gene on a plasmid but not with an
orf4
gene containing a site-directed mutation in the putative zinc metalloprotease motif. Although its mechanism of its action is being studied further, our results suggest that the Orf4 protein is a zinc metalloprotease that modulates the pathogenesis and reactogenicity of epidemic
V. cholerae
. Based on our findings, we name this VPI-encoded protein Mop (for modulation of pathogenesis).
Expression of Proinflammatory Cytokines and Receptors by Human Fallopian Tubes in Organ Culture following Challenge withNeisseria gonorrhoeae
Authors: Maisey Kevin , Nardocci Gino , Imarai Monica , Cardenas Hugo , Rios Miguel , Croxatto Horacio B. , Heckels John E. , Christodoulides Myron , Velasquez Luis A.
Published: December 2002

Infection of the Fallopian tubes (FT) byNeisseria gonorrhoeaecan lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor alpha (TNF-α) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.
Vibrio vulnificus
Induces Macrophage Apoptosis In Vitro and In Vivo
Authors: Kashimoto Takashige , Ueno Shunji , Hanajima Miyuki , Hayashi Hisae , Akeda Yukihiro , Miyoshi Shinichi , Hongo Toshiharu , Honda Takeshi , Susa Nobuyuki
Published: December 2002

In this study, we compared the apoptotic activities of clinical and environmental isolates of
Vibrio vulnificus
toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of
V. vulnificus
.
Campylobacter jejuniCytolethal Distending Toxin Promotes DNA Repair Responses in Normal Human Cells
Authors: Hassane Duane C. , Lee Robert B. , Pickett Carol L.
Published: December 2002

Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of theCampylobacter jejuni81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated withC. jejuniCDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G1phase of the cell cycle.
OxyR Acts as a Repressor of Catalase Expression in
Neisseria gonorrhoeae
Authors: Tseng Hsing-Ju , McEwan Alastair G. , Apicella Michael A. , Jennings Michael P.
Published: December 2002

It has been reported that
Neisseria gonorrhoeae
possesses a very high level of catalase activity, but the regulation of catalase expression has not been investigated extensively. In
Escherichia coli
and
Salmonella enterica
serovar Typhimurium, it has been demonstrated that OxyR is a positive regulator of hydrogen peroxide-inducible genes, including the gene encoding catalase. The
oxyR
gene from
N. gonorrhoeae
was cloned and used to complement an
E. coli oxyR
mutant, confirming its identity and function. The gene was inactivated by inserting a kanamycin resistance cassette and used to make a knockout allele on the chromosome of
N. gonorrhoeae
strain 1291. In contrast to
E. coli
, the
N. gonorrhoeae oxyR
::
kan
mutant expressed ninefold-more catalase activity and was more resistant to hydrogen peroxide killing than the wild type. These data are consistent with OxyR in
N. gonorrhoeae
acting as a repressor of catalase expression.
Feasibility of an HA2 Domain-Based Periodontitis Vaccine
Authors: DeCarlo A. A. , Huang Y. , Collyer C. A. , Langley D. B. , Katz J.
Published: December 2002

In a rat periodontitis model, preinoculation with the
Porphyromonas gingivalis
HA2 binding domain for hemoglobin provided protection from disease. Protection was associated with induced anti-HA2 immunoglobulin G (IgG) humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was administered in saline.
Induction of Emetic Response to Staphylococcal Enterotoxins in the House Musk Shrew (
Suncus murinus
)
Authors: Hu Dong-Liang , Omoe Katsuhiko , Shimoda Yu , Nakane Akio , Shinagawa Kunihiro
Published: December 2002

The emetic responses induced by staphylococcal enterotoxin A (SEA), SEB, SEC2, SED, SEE, SEG, SEH, and SEI in the house musk shrew (
Suncus murinus
) were investigated. SEA, SEE, and SEI showed higher emetic activity in the house musk shrew than the other SEs. SEB, SEC2, SED, SEG, and SEH also induced emetic responses in this animal model but relatively high doses were required. The house musk shrew appears to be a valuable model for studying the mechanisms of emetic reactions caused by SEs.
A Single-Copy Gene Encodes Kex1, a Serine Endoprotease of
Pneumocystis jiroveci
Authors: Kutty Geetha , Kovacs Joseph A.
Published: December 2002

We have cloned and characterized the
kex1
gene of
Pneumocystis jiroveci
. Unlike the case for
Pneumocystis carinii
, in which the homologous PRT-1 genes are multicopy,
kex1
is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus. Thus, substantial biological differences can be seen among
Pneumocystis
species.
Antibodies Present in Normal Human Serum Inhibit Invasion of Human Brain Microvascular Endothelial Cells byListeria monocytogenes
Authors: Hertzig Tobias , Weber Martin , Greiffenberg Lars , Holthausen Britta Schulte , Goebel Werner , Kim Kwang Sik , Kuhn Michael
Published: December 2002

Listeria monocytogenescauses meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms. The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of theinlBgene, the surface molecule InlB, which is a member of the internalin multigene family. In the present study we analyzed the role of the medium composition in the interaction ofL. monocytogeneswith HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum. The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells. The inhibitory component of human serum was identified as being associated withL. monocytogenes-specific antibodies present in the human serum. Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC byL. monocytogenes.
Inactivation of the
selB
Gene in
Methanococcus maripaludis:
Effect on Synthesis of Selenoproteins and Their Sulfur-Containing Homologs
Authors: Rother Michael , Mathes Isabella , Lottspeich Friedrich , Böck August
Published: December 2002

The genome of
Methanococcus maripaludis
harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated
selB
gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of
M. maripaludis
repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the
selB
mutation. Although the growth of
M. maripaludis
on H
2
/CO
2
is only marginally affected by the
selB
lesion, the gene is indispensable for growth on formate because
M. maripaludis
possesses only a selenocysteine-containing formate dehydrogenase.
Global Role for ClpP-Containing Proteases in Stationary-Phase Adaptation of
Escherichia coli
Authors: Weichart Dieter , Querfurth Nadine , Dreger Mathias , Hengge-Aronis Regine
Published: December 2002

To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the
clpA
,
clpX
, and
clpP
protease mutants of
Escherichia coli
were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the
clpA
,
clpX
, or
clpP
mutant failed to mount the growth-phase regulation found in the wild type. For example, in the
clpP
and
clpA
mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in
E. coli
are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.
Metal Ion Dependence of RecombinantEscherichia coliAllantoinase
Authors: Mulrooney Scott B. , Hausinger Robert P.
Published: December 2002

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. RecombinantEscherichia coliallantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess ∼1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (kcat= 34.7 min−1) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (kcatvalues of 5,000 and 28,200 min−1and 200 min−1, respectively). These rates and correspondingKmvalues (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significantKidifferences for the zinc and cobalt species (237 and 795 μM, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only theSisomer of allantoin, whereas the cobalt allantoinase prefers theSisomer, but also hydrolyzes theRisomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.
Cadaverine Inhibition of Porin Plays a Role in Cell Survival at Acidic pH
Authors: Samartzidou Hrissi , Mehrazin Mahsa , Xu Zhaohui , Benedik Michael J. , Delcour Anne H.
Published: December 2002

When grown at acidic pH,
Escherichia coli
cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.
Arsenite Oxidase
aox
Genes from a Metal-Resistant β-Proteobacterium
Authors: Muller Daniel , Lièvremont Didier , Simeonova Diliana Dancheva , Hubert Jean-Claude , Lett Marie-Claire
Published: December 2002

The β-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a
lacZ
-based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named
aoxA
and
aoxB
, as well as a putative promoter upstream of the
aoxA
gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by
aoxA
and
aoxB
share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of
Alcaligenes faecalis
, respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the
A. faecalis
enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by
aoxA
, strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.
Growth of Mycobacteria on Carbon Monoxide and Methanol
Authors: Park Sae W. , Hwang Eun H. , Park Hyuck , Kim Jeong A. , Heo Jinho , Lee Key H. , Song Taeksun , Kim Eungbin , Ro Young T. , Kim Si W. , Kim Young M.
Published: December 2002

Several mycobacterial strains, such asMycobacterium flavescens,Mycobacterium gastri,Mycobacterium neoaurum,Mycobacterium parafortuitum,Mycobacterium peregrinum,Mycobacterium phlei,Mycobacterium smegmatis,Mycobacterium tuberculosis, andMycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except forM. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH ofMycobacteriumsp. strain JC1 (formerlyAcinetobactersp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol.23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, exceptM. tuberculosis, grown on CO. The mycobacteria grown on methanol, except forM. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibitedN,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of theMycobacteriumsp. strain JC1 enzymes.
Protease-Deficient DegP Suppresses Lethal Effects of a Mutant OmpC Protein by Its Capture
Authors: CastilloKeller Maria , Misra Rajeev
Published: December 2002

The expression of assembly-defective outer membrane proteins can confer lethality if they are not degraded by envelope proteases. We report here that the expression of a mutant OmpC protein, OmpC
2Cys
, which forms disulfide bonds in the periplasm due to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP. This lethality is not observed in
dsbA
strains that have diminished ability to form periplasmic disulfide bonds. Our data show that this OmpC
2Cys
-mediated lethality in a
degP
::Km
r
dsbA
+
background can be reversed by a DegP variant, DegP
S210A
, that is devoid of its proteolytic activity but retains its reported chaperone activity. However, DegP
S210A
does not reverse the lethal effect of OmpC
2Cys
by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway. Displacement of OmpC
2Cys
by DegP
S210A
also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF.
Transcriptome Analysis of
Neisseria meningitidis
during Infection
Authors: Dietrich Guido , Kurz Sebastian , Hübner Claudia , Aepinus Christian , Theiss Stephanie , Guckenberger Matthias , Panzner Ursula , Weber Jacqueline , Frosch Matthias
Published: December 2002

Neisseria meningitidis
is the cause of septicemia and meningococcal meningitis. During the course of infection,
N
.
meningitidis
encounters multiple environments within its host, which makes rapid adaptation to environmental changes a crucial factor for neisserial pathogenicity. Employing oligonucleotide-based DNA microarrays, we analyzed the transcriptome of
N
.
meningitidis
during two key steps of meningococcal infection, i.e., the interaction with epithelial cells (HeLa cells) and endothelial cells (human brain microvascular endothelial cells). Seventy-two genes were differentially regulated after contact with epithelial cells, and 48 genes were differentially regulated after contact with endothelial cells, including a considerable proportion of well-known virulence genes. While a considerable number of genes were in concordance between bacteria adherent to both cell types, we identified several open reading frames that were differentially regulated in only one system. The data obtained with this novel approach may provide insight into the pathogenicity mechanisms of
N
.
meningitidis
and could demonstrate the importance of gene regulation on the transcriptional level during different stages of meningococcal infection.
Subunit Topology of Two 20S Proteasomes from
Haloferax volcanii
Authors: Kaczowka Steven J. , Maupin-Furlow Julie A.
Published: December 2002

Haloferax volcanii
, a halophilic archaeon, synthesizes three different proteins (α1, α2, and β) which are classified in the 20S proteasome superfamily. The α1 and β proteins alone form active 20S proteasomes; the role of α2, however, is not clear. To address this, α2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant
H. volcanii
. The α2 protein copurified with α1 and β in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described α1-β proteasome. Supplementing buffers with 10 mM CaCl
2
stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type
H. volcanii
synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the α1-β and α1-α2-β proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the α1-β proteasome was a symmetrical cylinder with α1 rings on each end. In contrast, the α1-α2-β proteasome appeared to be asymmetrical with homo-oligomeric α1 and α2 rings positioned on separate ends. Inter-α-subunit contacts were only detected when the ratio of α1 to α2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of α proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.
Biochemical and Mutational Characterization of the Heme Chaperone CcmE Reveals a Heme Binding Site
Authors: Enggist Elisabeth , Schneider Michael J. , Schulz Henk , Thöny-Meyer Linda
Published: December 2002

CcmE is a heme chaperone that binds heme transiently in the periplasm of
Escherichia coli
and delivers it to newly synthesized and exported
c
-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome
c
maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.
Transcriptional Organization of thePseudomonas putida tol-oprLGenes
Authors: Llamas María A. , Ramos Juan L. , Rodríguez-Herva José J.
Published: December 2002

Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. InPseudomonas putida, the seven genes,orf1,tolQ,tolR,tolA,tolB,oprL, andorf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterlesslacZgene revealed that the genes are arranged in two operons:orf1 tolQ tolR tolA tolBandoprL orf2. We were also able to find a transcript that was initiated at theorf1promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified theoprL orf2operon promoter within thetolBgene, and the −10 and −35 regions exhibited some similarity to those of σ70-recognized promoters. The transcription start point oforf1was located 91 bp upstream of theorf1start codon, and the −10/−35 region also exhibited σ70−10/−35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the β-galactosidase activities of different translational fusion constructs revealed that translation oftolAandorf2genes was dependent on the translation of their corresponding upstream genes (tolRandoprL, respectively).
Recruitment of MinC, an Inhibitor of Z-Ring Formation, to the Membrane in
Escherichia coli
: Role of MinD and MinE
Authors: Hu Zonglin , Saez Cristian , Lutkenhaus Joe
Published: December 2002

In
Escherichia coli
, the
min
system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.
Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium
Streptomyces coelicolor
A3(2)
Authors: Kaneko Masafumi , Ohnishi Yasuo , Horinouchi Sueharu
Published: December 2002

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium
Streptomyces coelicolor
A3(2). The recombinant enzyme, produced in
Escherichia coli
with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30°C, respectively. The

K
m

value for 4-coumarate and
k
cat
were determined as 131 ± 4 μM and 0.202 ± 0.007 s
−1
, respectively. The

K
m

value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (

K
m

, 190 ± 2 μM;
k
cat
, 0.475 ± 0.012 s
−1
), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for
S. coelicolor
A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala
294
and Ala
318
are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.
Requirement of ArcA for Redox Regulation in
Escherichia coli
under Microaerobic but Not Anaerobic or Aerobic Conditions
Authors: Alexeeva Svetlana , Hellingwerf Klaas J. , Teixeira de Mattos M. Joost
Published: December 2002

In
Escherichia coli,
the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type
E. coli
to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type
E. coli
and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome
o
- and
d
-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator.
Targeted Gene Disruption by Homologous Recombination in the Hyperthermophilic ArchaeonThermococcus kodakaraensisKOD1
Authors: Sato Takaaki , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: December 2002

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeonThermococcus kodakaraensisKOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5′-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of thetrpElocus in the host strain by homologous recombination, as disruption oftrpEwas expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring thepyrFmarker withintrpEwas constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl2method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-typepyrFinto the host chromosome attrpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between thepyrFmarker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtaintrpEdisruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.
Contrasting Sensitivities of
Escherichia coli
Aconitases A and B to Oxidation and Iron Depletion
Authors: Varghese Shery , Tang Yue , Imlay James A.
Published: December 2002

Superoxide damages dehydratases that contain catalytic [4Fe-4S]
2+
clusters. Aconitases are members of that enzyme family, and previous work showed that most aconitase activity is lost when
Escherichia coli
is exposed to superoxide stress. More recently it was determined that
E. coli
synthesizes at least two isozymes of aconitase, AcnA and AcnB. Synthesis of AcnA, the less-abundant enzyme, is positively controlled by SoxS, a protein that is activated in the presence of superoxide-generating chemicals. We have determined that this arrangement exists because AcnA is resistant to superoxide in vivo. Surprisingly, purified AcnA is extremely sensitive to superoxide and other chemical oxidants unless it is combined with an uncharacterized factor that is present in cell extracts. In contrast, AcnB is highly sensitive to a variety of chemical oxidants in vivo, in extracts, and in its purified form. Thus, the induction of AcnA during oxidative stress provides a mechanism to circumvent a block in the tricarboxylic acid cycle. AcnA appears to be as catalytically competent as AcnB, so the retention of the latter as the primary housekeeping enzyme must provide some other advantage. We observed that the [4Fe-4S] cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop. AcnA and other dehydratases do not show this trait. Demetallated AcnB is known to bind its cognate mRNA. The absence of AcnB activity also causes the accumulation and excretion of citrate, an iron chelator for which
E. coli
synthesizes a transport system. Thus, AcnB may be retained as the primary aconitase because the lability of its exposed cluster allows
E. coli
to sense and respond to iron depletion.
Multiple Promoter Inversions Generate Surface Antigenic Variation in
Mycoplasma penetrans
Authors: Horino Atsuko , Sasaki Yuko , Sasaki Tsuguo , Kenri Tsuyoshi
Published: December 2002

Mycoplasma penetrans
is a newly identified species of the genus
Mycoplasma
. It was first isolated from a urine sample from a human immunodeficiency virus (HIV)-infected patient.
M. penetrans
changes its surface antigen profile with high frequency. The changes originate from ON↔OFF phase variations of the P35 family of surface membrane lipoproteins. The P35 family lipoproteins are major antigens recognized by the human immune system during
M. penetrans
infection and are encoded by the
mpl
genes. Phase variations of P35 family lipoproteins occur at the transcriptional level of
mpl
genes; however, the precise genetic mechanisms are unknown. In this study, the molecular mechanisms of surface antigen profile change in
M. penetrans
were investigated. The focus was on the 46-kDa protein that is present in
M. penetrans
strain HF-2 but not in the type strain, GTU. The 46-kDa protein was the product of a previously reported
mpl
gene, pepIMP13, with an amino-terminal sequence identical to that of the P35 family lipoproteins. Nucleotide sequencing analysis of the pepIMP13 gene region revealed that the promoter-containing 135-bp DNA of this gene had the structure of an invertible element that functioned as a switch for gene expression. In addition, all of the
mpl
genes of
M. penetrans
HF-2 were identified using the whole-genome sequence data that has recently become available for this bacterium. There are at least 38
mpl
genes in the
M. penetrans
HF-2 genome. Interestingly, most of these
mpl
genes possess invertible promoter-like sequences, similar to those of the pepIMP13 gene promoter. A model for the generation of surface antigenic variation by multiple promoter inversions is proposed.