Identification of Salmonella enterica Serovar Typhimurium Genes Regulated during Biofilm Formation on Cholesterol Gallstone Surfaces

ABSTRACTSalmonellaspp. are able to form biofilms on abiotic and biotic surfaces.In vivostudies in our laboratory have shown thatSalmonellacan form biofilms on the surfaces of cholesterol gallstones in the gallbladders of mice and human carriers. Biofilm formation on gallstones has been demonstrated to be a mechanism of persistence. The purpose of this work was to identify and evaluateSalmonellasp. cholesterol-dependent biofilm factors. Differential gene expression analysis between biofilms on glass or cholesterol-coated surfaces and subsequent quantitative real-time PCR (qRT-PCR) revealed that type 1 fimbria structural genes and a gene encoding a putative outer membrane protein (ycfR) were specifically upregulated inSalmonella entericaserovar Typhimurium biofilms grown on cholesterol-coated surfaces. Spatiotemporal expression ofycfRand FimA verified their regulation during biofilm development on cholesterol-coated surfaces. Surprisingly, confocal and scanning electron microscopy demonstrated that a mutant of type 1 fimbria structural genes (ΔfimAICDHF) and aycfRmutant showed increased biofilm formation on cholesterol-coated surfaces.In vivoexperiments usingNramp1+/+mice harboring gallstones showed that only the ΔycfRmutant formed extensive biofilms on mouse gallstones at 7 and 21 days postinfection; ΔfimAICDHFwas not observed on gallstone surfaces after the 7-day-postinfection time point. These data suggest that inSalmonellaspp., wild-type type 1 fimbriae are important for attachment to and/or persistence on gallstones at later points of chronic infection, whereas YcfR may represent a specific potential natural inhibitor of initial biofilm formation on gallstones.

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Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

Journal of Bacteriology ◽

10.1128/jb.01620-09 ◽

2010 ◽

Vol 192 (12) ◽

pp. 2981-2990 ◽

Cited By ~ 65

Author(s):

Robert W. Crawford ◽

Kristin E. Reeve ◽

John S. Gunn

Keyword(s):

Salmonella Enterica ◽

Biofilm Formation ◽

Carrier State ◽

Enzyme Linked Immunosorbent Assay ◽

Cholesterol Gallstones ◽

Serovar Typhimurium ◽

Flagellar Proteins ◽

Coated Surfaces ◽

Biofilm Development ◽

Chronic Carrier

ABSTRACT The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.

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The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization

Infection and Immunity ◽

10.1128/iai.01135-15 ◽

2015 ◽

Vol 84 (1) ◽

pp. 375-384 ◽

Cited By ~ 26

Author(s):

Shalhevet Azriel ◽

Alina Goren ◽

Galia Rahav ◽

Ohad Gal-Mor

Keyword(s):

Salmonella Enterica ◽

Biofilm Formation ◽

Early Stage ◽

Response Regulator ◽

Stringent Response ◽

Negative Regulator ◽

Logarithmic Phase ◽

Content Type ◽

Serovar Typhimurium

Salmonella entericaserovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide.Salmonellapathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that inS.Typhimurium,dksAis induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates theSalmonellapathogenicity island 1 and motility-chemotaxis genes and is necessary forS.Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable forS.Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found thatdksAis spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infectionin vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulenceS.Typhimurium programs and therefore is a key virulence regulator ofSalmonella.

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SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

Infection and Immunity ◽

10.1128/iai.05592-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4342-4352 ◽

Cited By ~ 52

Author(s):

Dhaarini Raghunathan ◽

Timothy J. Wells ◽

Faye C. Morris ◽

Robert K. Shaw ◽

Saeeda Bobat ◽

...

Keyword(s):

Salmonella Enterica ◽

Biofilm Formation ◽

Protective Antigen ◽

Outer Membrane Proteins ◽

Cell Aggregation ◽

Sub Saharan Africa ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTSalmonella entericais a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins ofSalmonellaare of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin ofSalmonella entericaserovar Typhimurium, were examined. We demonstrated that SadA is exposed on theSalmonellacell surfacein vitroandin vivoduring infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection againstSalmonella.

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Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00419-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 1

Author(s):

Edna M. Ondari ◽

Jennifer N. Heath ◽

Elizabeth J. Klemm ◽

Gemma Langridge ◽

Lars Barquist ◽

...

Keyword(s):

Salmonella Enterica ◽

Protein Translocation ◽

Invasive Disease ◽

Immune Serum ◽

Insertion Mutant ◽

Content Type ◽

Serovar Typhimurium ◽

Serum Killing ◽

Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

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Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

Infection and Immunity ◽

10.1128/iai.00211-18 ◽

2018 ◽

Vol 86 (9) ◽

Author(s):

Vivek Belde ◽

Matthew P. Cravens ◽

Dania Gulandijany ◽

Justin A. Walker ◽

Isabel Palomo-Caturla ◽

...

Keyword(s):

Antibody Response ◽

B Cell ◽

Salmonella Enterica ◽

Passive Immunization ◽

Terminal Deoxynucleotidyl Transferase ◽

Human Infants ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

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Clarithromycin Exerts an Antibiofilm Effect against Salmonella enterica Serovar Typhimurium rdar Biofilm Formation and Transforms the Physiology towards an Apparent Oxygen-Depleted Energy and Carbon Metabolism

Infection and Immunity ◽

10.1128/iai.00510-20 ◽

2020 ◽

Vol 88 (11) ◽

Author(s):

Munirah Zafar ◽

Humera Jahan ◽

Sulman Shafeeq ◽

Manfred Nimtz ◽

Lothar Jänsch ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Salmonella Enterica ◽

Biofilm Formation ◽

Antibiofilm Activity ◽

Bacterial Cells ◽

Content Type ◽

Regulatory Pathways ◽

Heat Shock Stress ◽

Extracellular Matrix Components ◽

Serovar Typhimurium

ABSTRACT Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection toward various forms of environmental stresses to individual cells. Thus, bacterial cells within biofilms become tolerant against antimicrobials and the immune system. In the present study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry, and rough) biofilm formation of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium ATCC 14028 (Nalr) at a 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment, suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be concomitantly downregulated. Although fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifted the cells toward an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation was downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and l-arginine catabolism, potentially also to prevent cytosolic acidification, was upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

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Facultative Anaerobes Shape Multispecies Biofilms Composed of Meat Processing Surface Bacteria and Escherichia coli O157:H7 or Salmonella enterica Serovar Typhimurium

Applied and Environmental Microbiology ◽

10.1128/aem.01123-19 ◽

2019 ◽

Vol 85 (17) ◽

Author(s):

Jeyachchandran Visvalingam ◽

Hui Wang ◽

Tim C. Ells ◽

Xianqin Yang

Keyword(s):

Food Processing ◽

Salmonella Enterica ◽

Biofilm Formation ◽

Single Species ◽

Meat Processing ◽

Content Type ◽

E Coli ◽

Facultative Anaerobes ◽

Serovar Typhimurium ◽

Multispecies Biofilms

ABSTRACT This study investigated the microbial dynamics in multispecies biofilms of Escherichia coli O157:H7 strain 1934 (O157) or Salmonella enterica serovar Typhimurium ATCC 14028 (ST) and 40 strains of meat processing surface bacteria (MPB). Biofilms of O157 or ST with/without MPB were developed on stainless steel coupons at 15°C for up to 6 days. Bacteria in suspensions (inoculum, days 2 and 6) and biofilms (days 2 and 6) were enumerated by plating. The composition of multispecies cultures was determined by 16S rRNA gene sequencing. In suspensions, levels of O157 and ST were ∼2 log higher in single-species than in multispecies cultures on both sampling days. ST was 3 log higher in single-species than in multispecies biofilms. A similar trend, though to a lesser extent, was observed for O157 in biofilms on day 2 but not on day 6. No difference (P > 0.05) in bacterial counts was noted for the two MPB-pathogen cocultures at any time during incubation. Bacterial diversity in multispecies cultures decreased with incubation time, irrespective of the pathogen or culture type. The changes in the relative abundance of MPB were similar for the two MPB-pathogen cocultures, though different interbacterial interactions were noted. Respective fractions of ST and O157 were 2.1% and 0.97% initially and then 0.10% and 0.07% on day 2, and 0.60% and 0.04% on day 6. The relative proportions of facultative anaerobes in both multispecies cultures were greater in both suspensions and biofilms than in the inoculum. Citrobacter, Hafnia, Aeromonas, and Carnobacterium predominated in biofilms but not always in the planktonic cultures. IMPORTANCE Results of this study demonstrate that Salmonella enterica serovar Typhimurium and E. coli O157:H7 can integrate into biofilms when cocultured with bacteria from meat plant processing surfaces. However, the degree of biofilm formation for both pathogens was substantially reduced in the presence of the competing microbiota, with S. Typhimurium more greatly affected than E. coli O157:H7. The expression of extracellular determinants such as curli and cellulose appears to be less important for biofilm formation of the pathogens in multispecies cultures than in monoculture. In contrast to previous reports regarding food processing surface bacteria, data collected here also demonstrate that facultative anaerobes may have a competitive edge over strict aerobes in establishing multispecies biofilms. It would be important to take into account the presence of background bacteria when evaluating the potential persistence of a pathogen in food processing facilities.

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The Histidine Kinase BinK Is a Negative Regulator of Biofilm Formation and Squid Colonization

Journal of Bacteriology ◽

10.1128/jb.00037-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2596-2607 ◽

Cited By ~ 21

Author(s):

John F. Brooks ◽

Mark J. Mandel

Keyword(s):

Histidine Kinase ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Negative Regulator ◽

Epithelial Tissue ◽

Temperature Modulation ◽

Content Type ◽

Animal Hosts ◽

Biofilm Development

ABSTRACTBacterial colonization of animal epithelial tissue is a dynamic process that relies on precise molecular communication. Colonization ofEuprymna scolopesbobtail squid byVibrio fischeribacteria requires bacterial aggregation in host mucus as the symbiont transitions from a planktonic lifestyle in seawater to a biofilm-associated state in the host. We have identified a gene,binK(biofilm inhibitor kinase; VF_A0360), which encodes an orphan hybrid histidine kinase that negatively regulates theV. fischerisymbiotic biofilm (Syp)in vivoandin vitro. We identifiedbinKmutants as exhibiting a colonization advantage in a global genetic screen, a phenotype that we confirmed in controlled competition experiments. Bacterial biofilm aggregates in the host are larger in strains lacking BinK, whereas overexpression of BinK suppresses biofilm formation and squid colonization. Signaling through BinK is required for temperature modulation of biofilm formation at 28°C. Furthermore, we present evidence that BinK acts upstream of SypG, the σ54-dependent transcriptional regulator of thesypbiofilm locus. The BinK effects are dependent on intact signaling in the RscS-Syp biofilm pathway. Therefore, we propose that BinK antagonizes the signal from RscS and serves as an integral component inV. fischeribiofilm regulation.IMPORTANCEBacterial lifestyle transitions underlie the colonization of animal hosts from environmental reservoirs. Formation of matrix-enclosed, surface-associated aggregates (biofilms) is common in beneficial and pathogenic associations, but investigating the genetic basis of biofilm development in live animal hosts remains a significant challenge. Using the bobtail squid light organ as a model, we analyzed putative colonization factors and identified a histidine kinase that negatively regulates biofilm formation at the host interface. This work reveals a novelin vivobiofilm regulator that influences the transition of bacteria from their planktonic state in seawater to tight aggregates of cells in the host. The study enriches our understanding of biofilm regulation and beneficial colonization by an animal's microbiome.

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Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

Infection and Immunity ◽

10.1128/iai.01304-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 221-232 ◽

Cited By ~ 16

Author(s):

Cristina Núñez-Hernández ◽

Ana Alonso ◽

M. Graciela Pucciarelli ◽

Josep Casadesús ◽

Francisco García-del Portillo

Keyword(s):

Salmonella Enterica ◽

Pathogenicity Island ◽

Effector Proteins ◽

Intracellular Bacteria ◽

Secretion Systems ◽

Cultured Fibroblasts ◽

Content Type ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium

ABSTRACTSalmonella entericauses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recentin vivostudies have shown that intracellular bacteria activate the TTSS encoded bySalmonellapathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observedin vivoin bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that theS. entericaserovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

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Differential Gene Expression Patterns ofYersinia pestisandYersinia pseudotuberculosisduring Infection and Biofilm Formation in the Flea Digestive Tract

mSystems ◽

10.1128/msystems.00217-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 8

Author(s):

Iman Chouikha ◽

Daniel E. Sturdevant ◽

Clayton Jarrett ◽

Yi-Cheng Sun ◽

B. Joseph Hinnebusch

Keyword(s):

Digestive Tract ◽

Biofilm Formation ◽

Yersinia Pseudotuberculosis ◽

Expression Patterns ◽

Type Vi Secretion System ◽

Genetic Changes ◽

Content Type ◽

Biofilm Development

ABSTRACTYersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in theYersinia pseudotuberculosisprogenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performedin vivotranscriptional profiling ofY. pestis, aY. pseudotuberculosiswild-type strain (unable to form biofilm in the flea foregut), and aY. pseudotuberculosismutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, theY. pseudotuberculosismutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of thehmsgenes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. TheY. pseudotuberculosismutant uniquely expressed much higher levels ofYersiniatype VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage byY. pseudotuberculosisbut not for blockage byY. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection.IMPORTANCEY. pestisemerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes fromY. pseudotuberculosis. Parallel comparisons of thein vitroandin vivotranscriptomes ofY. pestisand twoY. pseudotuberculosisvariants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into howY. pestishas adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

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