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Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated CO 2 , and the trans -acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH , a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis . In the well-studied B. subtilis system, σ H is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH -null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. σ H positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of σ H and AbrB on the expression of atxA .

Summary The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

Cholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria. Bacillus anthracis produces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions. B. cereus and B. thuringiensis , species closely related to B. anthracis , produce CDCs with significant amino acid sequence homology to ALO. Transcription of the B. cereus and B. thuringiensis CDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from the papR gene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes the B. anthracis alo gene, and the B. anthracis genome contains three plcR -like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA of alo , papR , and the three plcR -like genes in spleens of B. anthracis -infected mice, indicating gene expression in vivo. Analysis of alo transcription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels of alo transcripts and ALO protein were unaffected by deletion of papR or disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally active plcR and papR genes in B. anthracis and a PlcR box in the promoter region of the alo gene, alo expression is independent of this control system.

The poly- d -glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO 2 /bicarbonate signal and three plasmid-borne regulators: atxA , acpA , and acpB . Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB , two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO 2 compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO 2 . In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO 2 . The 5′-end mapping of gene transcripts revealed atxA -regulated and atxA -independent apparent transcription start sites for capB , acpA , and acpB . Transcripts mapping to all atxA -regulated start sites were increased during growth in elevated CO 2 . The acpA gene has one atxA -regulated and one atxA -independent start site. acpB lies downstream of capBCAD . A single atxA -independent start site maps immediately upstream of acpB. atxA -mediated control of acpB appears to occur via transcriptional read-through from atxA -dependent start sites 5′ of capB . One atxA -independent and two atxA -regulated start sites map upstream of capB . Transcription from the atxA- regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB , in agreement with the current model for epistatic relationships between the regulators.

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region. They are expressed on the surface of B. anthracis. The A regions of the two proteins were predicted to adopt similar structural folds as CNA. Circular dichroism analysis of the recombinant A regions of the two proteins (rBA0871A and rBA5258A) indicate that their secondary structure compositions are similar to those of the A regions of CNA and other cell wall-anchored adhesins. We demonstrate through solid phase binding assays and surface plasmon resonance analyses that rBA0871A and rBA5258A specifically bound type I collagen in a dose-dependent and saturable manner. Their dissociation constants (KD) for collagen are 1.6-3.2 microm for rBA0871A and 0.6-0.9 microm for rBA5258A, respectively. We further demonstrate that BA0871 and BA5258 can mediate cell attachment to collagen when expressed on the surface of a heterologous host bacterium. To our knowledge these are the first two adhesins of B. anthracis described, which may have important implications for our understanding of the pathogenic mechanisms explored by this organism.

Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD(50) and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis , Bacillus cereus , and Bacillus subtilis . The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5′ and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

This project provides a proof-of-principle technology demonstration for SDMS, where a distributed suite of sensors is integrated with active components and passive structures of types expected to be encountered in next generation nuclear power reactor and plant systems. The project employs state-of-the-art operational sensors, advanced stressor-based instrumentation, distributed computing, RF data network modules and signal processing to improve the monitoring and assessment of the power reactor system and gives data that is used to provide prognostics capabilities.

Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis , has been associated with two regulatory genes, atxA and acpA , located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1 + pXO2 + parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA -regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA . In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA -activated capsule gene transcription is not acpA dependent. The newly discovered atxA -regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis , previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA -regulated genes should be investigated.

Section 203 of Executive Order 13123 requires all Federal agencies to reduce energy consumption. While there is an exemption for Federal laboratories and research and development facilities, industrial facilities do not fall under this program. The goal of this task was to identify and characterize key industrial Federal sites to assist in establishment of a support program for energy reduction in Federal industrial facilities. This document identifies key industrial sites in both the Defense Department and civilian agencies that could be prime candidates for energy-efficiency improvements. For these targeted agencies, location, gross square footage, and site energy consumption, cost and general industrial process information is provided (if available).

The management of the Spent Nuclear Fuel (SNF) project at the Hanford K-Basin in the 100 N Area has successfully restructured the preventive maintenance, spare parts inventory requirements, and the operator rounds data requirements. In this investigation, they continue to examine the different facets of the operations and maintenance (O&M) of the K-Basin cleanup project in search of additional reliability and cost savings. This report focuses on the initial findings of a team of PNNL engineers engaged to identify potential opportunities for reducing the cost of O&M through the application of advanced diagnostics (fault determination) and prognostics (residual life/reliability determination). The objective is to introduce predictive technologies to eliminate or reduce high impact equipment failures. The PNNL team in conjunction with the SNF engineers found the following major opportunities for cost reduction and/or enhancing reliability: (1) Provide data routing and automated analysis from existing detection systems to a display center that will engage the operations and engineering team. This display will be operator intuitive with system alarms and integrated diagnostic capability. (2) Change operating methods to reduce major transients induced in critical equipment. This would reduce stress levels on critical equipment. (3) Install a limited sensor set on failure pronemore » critical equipment to allow degradation or stressor levels to be monitored and alarmed. This would provide operators and engineers with advance guidance and warning of failure events. Specific methods for implementation of the above improvement opportunities are provided in the recommendations. They include an Integrated Water Treatment System (IWTS) decision support system, introduction of variable frequency drives on certain pump motors, and the addition of limited diagnostic instrumentation on specified critical equipment.« less

The Bacillus anthracis genome consists of an approximately 5.3-Mb chromosome and two plasmids, pXO1 (182 kb) and pXO2 (96 kb). Genetic analysis has focused primarily on the structural genes for the anthrax toxin proteins, pagA, lef, and cya, the biosynthetic genes for capsule synthesis, capB, capC, and capA, and a gene associated with depolymerization of capsule, dep. The three toxin genes are located at distinct loci on pXO1, while the cap and dep genes are arranged in an apparent operon on pXO2. Additional genes that may play a role in B. anthracis virulence include the germination operon gerX and the general stress transcription factor sigB. Host-related signals affecting transcription of the toxin and capsule genes include temperature (37°C) and bicarbonate/CO2. The B. anthracis plasmids carry two regulatory genes that share little sequence similarity with regulators in other bacteria. The pXO1-encoded gene atxA positively controls expression of the toxin and capsule genes, and has been implicated in control of other genes of unknown function. atxA mutants are avirulent in mice, and mice infected with atxA-null strains show a decreased immunological response to the toxin proteins. The pXO2-encoded regulator, acpA, shares sequence similarity with atxA. Yet acpA function appears to be restricted to positive control of capsule gene expression. The chromosomal gene abrB, a homologue of a well-characterized B. subtilis transition state regulator, controls growth phase-specific transcription of the toxin genes. Genetic manipulation of B. anthracis can be achieved by using natural means of DNA transfer and by electroporation of recombinant DNAs into B. anthracis. Genetic exchange can occur between B. anthracis strains and between B. anthracis and closely-related species. Although pXO1 and pXO2 are not self-transmissible, these plasmids and others can be transferred by conjugative plasmids originating in B. thuringiensis. Generalized transducing phage that permit inter-species transfer of chromosomal and plasmid DNA have also been described.

This document provides information that will be useful to FEMP in targeting and identifying Federal sites for energy efficiency project potential. The document consolidates Federal energy data into a single source reference from which FEMP can work to more efficiently target opportunities.

This paper is the result of an extensive literature review and technology evaluation, performed to determine the status of sensors and measurement technologies.

Bacillus anthracis plasmids pX01 and pX02, harboured by the Sterne and Pasteur strains, respectively, have been sequenced by random 'shotgun' cloning and high throughout sequence analysis. These sequences have been assembled (Sequencher) to generate a circulate pX01 plasmid containing 181 656 bp and a single linear (gapped) pX02 contig containing at least 93.479 bp. Initial annotation suggests that the two plasmids combined contain at least 200 potential open reading frames (ORFs) with < 40% having significant similarity to sequences registered in open databases. Collectively, only 118 566 bp of the pX01 DNA (65%) represent predicted coding regions. This value is similar to published gene densities for other plasmids and is indicative of the larger intergenic spaces in plasmids vs those found in the chromosomes of the parental microbes (85-93% gene density). A 70 kbp region including the toxin genes (cya, lef and pag) is distinct from the remainder of the pX01 sequence: (1) it has a lower gene density (58 vs 70%) than the remaining 111 kbp; (2) it contains all but one of the co-regulated transcriptional fusions identified by transposon mutagenesis (Hoffmaster & Koehler 1997) and (3) it contains a significantly higher proportion of positive BLAST scores (62 vs 20%) for putative ORFs. These data suggest different origins for the two regions of pX01.

Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a transactivator of anthrax toxin synthesis. Here we show that transcription of atxA does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an omega km-2 cassette. Transcription of all three toxin genes is decreased in the absence of atxA. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the atxA-null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the P1 site is sufficient for atxA-mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5' ends mapping to these sites are not detected in the atxA-null mutant. The atxA-null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atxA-null mutant-infected mice. These data suggest that the atxA gene product also regulates toxin gene expression during infection.

The pag gene of Bacillus anthracis, located on plasmid pXO1 (185 kb), encodes protective antigen, a component of the anthrax lethal and edema toxins. Synthesis of protective antigen is enhanced during growth of the organism with elevated levels of CO2. The CO2 effect is at the level of transcription, and pXO1-encoded regulatory factors have been implicated in control of pag expression. We used a Tn917-LTV3 insertion mutant of B. anthracis in which the wild-type pag gene on pXO1 was replaced with a pag-lacZ transcriptional fusion to monitor pag promoter activity. Expression of the pag-lacZ fusion is induced five- to eightfold during growth in 5% CO2 compared with growth in air. Growth in 20% CO2 increases transcription up to 19-fold. By monitoring pag-lacZ expression in atmospheres with different O2 and CO2 concentrations, we demonstrated definitively that the CO2 effect is specific and not simply a result of increased anaerobiosis. The results of 5' end mapping of pag transcripts indicate multiple sites of transcript initiation. We have determined two major apparent start sites, designated P1 and P2, located at positions -58 and -26 relative to the translation initiation codon, respectively. Analysis of total RNA from late-log-phase cells shows comparable initiation from P1 and P2 in wild-type strains grown in aerobic conditions. However, initiation from P1 is increased approximately 10-fold in cultures grown with an elevated level (5%) of CO2. We have identified a locus on pXO1, more than 13 kb upstream from the pag gene, which enhances pag transcription. When added in trans, this locus increases the level of transcripts with 5' ends mapping to P1 but has no effect on the level of transcripts with 5' ends mapping to P2. The CO2 effect on P1 is observed only in the presence of the activator locus.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.

Virulent and certain avirulent strains of Bacillus anthracis harbor a plasmid, designated pXO2, which is involved in the synthesis of capsules. Two classes of rough, noncapsulated (Cap-) variants were isolated from the capsule-producing (Cap+) Pasteur vaccine strains ATCC 6602 and ATCC 4229. One class was cured of pXO2, and the other class still carried it. Reversion to Cap+ was demonstrable only in rough variants which had retained pXO2. Proof that pXO2 is involved in capsule synthesis came from experiments in which the plasmid was transferred by CP-51-mediated transduction and by a mating system in which plasmid transfer is mediated by a Bacillus thuringiensis fertility plasmid, pXO12. Cells of Bacillus cereus and a previously noncapsulated (pXO2-) strain of B. anthracis produced capsules after the acquisition of pXO2.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading., Author Summary Bacillus thuringiensis (Bt) is a well known entomopathogenic bacterium successfully used as a biopesticide for fifty years. The insecticidal properties of Bt are mainly due to specific toxins forming a crystal inclusion associated with the spore. After ingestion by susceptible insect larvae, toxins could induce favorable conditions for spore germination. The bacteria multiply in the insect and coordinate their behavior using signaling molecules involved in quorum sensing. The activation of the quorum sensor PlcR leads to the production of virulence factors allowing the bacteria to kill the insect host. Here we show that, in the cadaver, Bt shifts from a virulent to a necrotrophic lifestyle during which a second quorum sensor (NprR) becomes functional. NprR activates genes encoding degradative enzymes (proteases, lipases and chitinases) and a lipopeptide (kurstakin) involved in swarming and biofilm formation. The kurstakin is also essential for the survival of Bt after insect death. This suggests that NprR allows the bacteria to survive and eventually to sporulate in the host cadaver, thus improving their ability to disseminate in the environment. Altogether these results show that the pathogenic and necrotrophic lifestyles of Bt are tightly controlled by two quorum-sensing systems acting sequentially during the infection process.