Oxidative stress is intrinsic to staphylococcal adaptation to fatty acid synthesis antibiotics.

Antibiotics inhibiting the fatty acid synthesis pathway (FASII) of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed the consequences and effectors of FASII-antibiotic (anti-FASII) adaptation. Anti-FASII induced lasting expression changes without genomic rearrangements. Several identified regulators affected the timing of adaptation outgrowth. Adaptation resulted in decreased expression of major virulence factors. Conversely, stress responses were globally increased and adapted bacteria were more resistant to peroxide killing. Importantly, pre-exposure to peroxide led to faster anti-FASII-adaptation by stimulating eFA incorporation. This adaptation differs from reports of peroxide-stimulated antibiotic efflux, which leads to tolerance. In vivo, anti-FASII-adapted S. aureus killed the insect host more slowly but continued multiplying. We conclude that staphylococcal adaptation to FASII antibiotics involves reprogramming, which decreases virulence and increases stress resistance. Peroxide, produced by the host to combat infection, favors anti-FASII adaptation.

FabT, a Bacterial Transcriptional Repressor That Limits Futile Fatty Acid Biosynthesis.

Phospholipids are vital membrane constituents that determine cell functions and interactions with the environment. For bacterial pathogens, rapid adjustment of phospholipid composition to changing conditions during infection can be crucial for growth and survival. Fatty acid synthesis (FASII) regulators are central to this process. This review puts the spotlight on FabT, a MarR-family regulator of FASII characterized in streptococci, enterococci, and lactococci. Roles of FabT in virulence, as reported in mouse and nonhuman primate infection models, will be discussed. We present FabT structure, the FabT regulon, and changes in FabT regulation according to growth conditions. A unique feature of FabT concerns its modulation by an unconventional corepressor, acyl-acyl-carrier protein (ACP). Some bacteria express two ACP proteins, which are distinguished by their interactions with endogenous or exogenous fatty acid sources, one of which causes strong FabT repression. This system seems to allow preferred use of environmental fatty acids, thereby saving energy by limiting futile FASII activity. Control of fabT expression and FabT activity link various metabolic pathways to FASII. The various physiological consequences of FabT loss summarized here suggest that FabT has potential as a narrow range therapeutic target.

Mechanisms of Acetoin Toxicity and Adaptive Responses in an Acetoin-Producing Species, Lactococcus lactis.

Acetoin, 3-hydroxyl,2-butanone, is extensively used as a flavor additive in food products. This volatile compound is produced by the dairy bacterium Lactococcus lactis when aerobic respiration is activated by haem addition, and comprises ∼70% of carbohydrate degradation products. Here we investigate the targets of acetoin toxicity, and determine how acetoin impacts L. lactis physiology and survival. Acetoin caused damage to DNA and proteins, which related to reactivity of its keto group. Acetoin stress was reflected in proteome profiles, which revealed changes in lipid metabolic proteins. Acetoin provoked marked changes in fatty acid composition, with massive accumulation of cycC19:0 cyclopropane fatty acid at the expense of its unsaturated C18:1 fatty acid precursor. Deletion of the cfa gene, encoding the cycC19:0 synthase, sensitized cells to acetoin stress. Acetoin-resistant transposon mutagenesis revealed a hot spot in the high affinity phosphate transporter operon pstABCDEF, which is known to increase resistance to multiple stresses. This work reveals the causes and consequences of acetoin stress on L. lactis, and may facilitate control of lactic acid bacteria production in technological processes. IMPORTANCE Acetoin, 3-hydroxyl,2-butanone, has diverse uses in chemical industry, agriculture, and dairy industries as a volatile compound that generates aromas. In bacteria, it can be produced in high amount by Lactococcus lactis when it grows under aerobic respiration. However, acetoin production can be toxic and detrimental for growth and/or survival. Our results showed that it damages DNA and proteins via its keto group. We also showed that acetoin modifies membrane fatty acid composition with the production of cyclopropane C19:0 fatty acid at the expense of an unsaturated C18:1. We isolated mutants more resistant to acetoin than the wild-type strain. All of them mapped to a single locus pstABCDEF operon, suggesting a simple means to limit acetoin toxicity in dairy bacteria and to improve its production.

Type II Fatty Acid Synthesis Pathway and Cyclopropane Ring Formation Are Dispensable during Enterococcus faecalis Systemic Infection.

Enterococcus faecalis, a multiple antibiotic-resistant Gram-positive bacterium, has emerged as a serious nosocomial pathogen. Here, we used a genetic approach to characterize the strategies used by E. faecalis to fulfill its requirements for endogenous fatty acid (FA) synthesis in vitro and in vivo. The type II fatty acid synthesis (FASII) pathway is encoded by two operons and two monocistronic genes. Expression of all of these genes is repressed by exogenous FAs, which are incorporated into the E. faecalis membrane and modify its composition. Deletion of nine genes of the 12-gene operon abolished growth in an FA-free medium. Addition of serum, which is lipid rich, restored growth. Interestingly, the E. faecalis membrane contains cyclic fatty acids that modify membrane properties but that are unavailable in host serum. The cfa gene that encodes the cyclopropanation process is located in a locus independent of the FASII genes. Its deletion did not alter growth under the conditions tested, but yielded bacteria devoid of cyclic FAs. No differences were observed between mice infected with wild-type (WT) or with FASII or cyclopropanation mutant strains, in terms of bacterial loads in blood, liver, spleen, or kidneys. We conclude that in E. faecalis, neither FASII nor cyclopropanation enzymes are suitable antibiotic targets. IMPORTANCE Membrane lipid homeostasis is crucial for bacterial physiology, adaptation, and virulence. Fatty acids are constituents of the phospholipids that are essential membrane components. Most bacteria incorporate exogenous fatty acids into their membranes. Enterococcus faecalis has emerged as a serious nosocomial pathogen that is responsible for urinary tract infections, bacteremia, and endocarditis and is intrinsically resistant to numerous antibiotics. E. faecalis synthesizes saturated and unsaturated fatty acids, as well as cyclic fatty acids that are not found in the human host. Here, we characterized mutant strains deficient in fatty acid synthesis and modification using genetic, biochemical, and in vivo approaches. We conclude that neither the fatty acid synthesis pathway nor the cyclopropanation enzyme are suitable targets for E. faecalis antibiotic development.

Do Primocolonizing Bacteria Enable Bacteroides thetaiotaomicron Intestinal Colonization Independently of the Capacity To Consume Oxygen?

Aerobic bacteria are frequent primocolonizers of the human naive intestine. Their generally accepted role is to eliminate oxygen, which would allow colonization by anaerobes that subsequently dominate bacterial gut populations. In this hypothesis-based study, we revisited this dogma experimentally in a germfree mouse model as a mimic of the germfree newborn. We varied conditions leading to the establishment of the dominant intestinal anaerobe Bacteroides thetaiotaomicron Two variables were introduced: Bacteroides inoculum size and preestablishment by bacteria capable or not of consuming oxygen. High Bacteroides inoculum size enabled its primocolonization. At low inocula, we show that bacterial preestablishment was decisive for subsequent Bacteroides colonization. However, even non-oxygen-respiring bacteria, a hemAEscherichia coli mutant and the intestinal obligate anaerobe Clostridium scindens, facilitated Bacteroides establishment. These findings, which are supported by recent reports, revise the long-held assumption that oxygen scavenging is the main role for aerobic primocolonizing bacteria. Instead, we suggest that better survival of aerobic bacteria ex vivo during vectorization between hosts could be a reason for their frequent primocolonization.

(p)ppGpp/GTP and Malonyl-CoA Modulate Staphylococcus aureus Adaptation to FASII Antibiotics and Provide a Basis for Synergistic Bi-Therapy.

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.

Permissive Fatty Acid Incorporation Promotes Staphylococcal Adaptation to FASII Antibiotics in Host Environments.

The essentiality of fatty acid synthesis (FASII) products in the human pathogen Staphylococcus aureus is the underlying rationale for FASII-targeted antimicrobial drug design. Reports of anti-FASII efficacy in animals support this choice. However, restricted test conditions used previously led us to investigate this postulate in a broader, host-relevant context. We report that S. aureus rapidly adapts to FASII antibiotics without FASII mutations when exposed to host environments. FASII antibiotic administration upon signs of infection, rather than just after inoculation as commonly practiced, fails to eliminate S. aureus in a septicemia model. In vitro, serum lowers S. aureus membrane stress, leading to a greater retention of the substrates required for environmental fatty acid (eFA) utilization: eFAs and the acyl carrier protein. In this condition, eFA occupies both phospholipid positions, regardless of anti-FASII selection. Our results identify S. aureus membrane plasticity in host environments as a main limitation for using FASII antibiotics in monotherapeutic treatments.

Genetics of Lactococci.

Lactococcus lactis is the best characterized species among the lactococci, and among the most consumed food-fermenting bacteria worldwide. Thanks to their importance in industrialized food production, lactococci are among the lead bacteria understood for fundamental metabolic pathways that dictate growth and survival properties. Interestingly, lactococci belong to the Streptococcaceae family, which includes food, commensal and virulent species. As basic metabolic pathways (e.g., respiration, metal homeostasis, nucleotide metabolism) are now understood to underlie virulence, processes elucidated in lactococci could be important for understanding pathogen fitness and synergy between bacteria. This chapter highlights major findings in lactococci and related bacteria, and covers five themes: distinguishing features of lactococci, metabolic capacities including the less known respiration metabolism in Streptococcaceae, factors and pathways modulating stress response and fitness, interbacterial dialogue via metabolites, and novel applications in health and biotechnology.

Task Distribution between Acetate and Acetoin Pathways To Prolong Growth in Lactococcus lactis under Respiration Conditions.

Lactococcus lactis is the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability.IMPORTANCELactococcus lactis is used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.

Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.

Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, in vitro, and animal studies report treatment failure, notably against Staphylococcus aureus The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies.

The Staphylococcus aureus FASII bypass escape route from FASII inhibitors.

Antimicrobials targeting the fatty acid synthesis (FASII) pathway are being developed as alternative treatments for bacterial infections. Emergence of resistance to FASII inhibitors was mainly considered as a consequence of mutations in the FASII target genes. However, an alternative and efficient anti-FASII resistance strategy, called here FASII bypass, was uncovered. Bacteria that bypass FASII incorporate exogenous fatty acids in membrane lipids, and thus dispense with the need for FASII. This strategy is used by numerous Gram-positive low GC % bacteria, including streptococci, enterococci, and staphylococci. Some bacteria repress FASII genes once fatty acids are available, and "constitutively" shift to FASII bypass. Others, such as the major pathogen Staphylococcus aureus, can undergo high frequency mutations that favor FASII bypass. This capacity is particularly relevant during infection, as the host supplies the fatty acids needed for bacteria to bypass FASII and thus become resistant to FASII inhibitors. Screenings for anti-FASII resistance in the presence of exogenous fatty acids confirmed that FASII bypass confers anti-FASII resistance among clinical and veterinary isolates. Polymorphisms in S. aureus FASII initiation enzymes favor FASII bypass, possibly by increasing availability of acyl-carrier protein, a required intermediate. Here we review FASII bypass and consequences in light of proposed uses of anti-FASII to treat infections, with a focus on FASII bypass in S. aureus.

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus.

The need for new antimicrobials to treat bacterial infections has led to the use of type II fatty acid synthesis (FASII) enzymes as front-line targets. However, recent studies suggest that FASII inhibitors may not work against the opportunist pathogen Staphylococcus aureus, as environmental fatty acids favor emergence of multi-anti-FASII resistance. As fatty acids are abundant in the host and one FASII inhibitor, triclosan, is widespread, we investigated whether fatty acid pools impact resistance in clinical and veterinary S. aureus isolates. Simple addition of fatty acids to the screening medium led to a 50% increase in triclosan resistance, as tested in 700 isolates. Moreover, nonculturable triclosan-resistant fatty acid auxotrophs, which escape detection under routine conditions, were uncovered in primary patient samples. FASII bypass in selected isolates correlated with polymorphisms in the acc and fabD loci. We conclude that fatty-acid-dependent strategies to escape FASII inhibition are common among S. aureus isolates and correlate with anti-FASII resistance and emergence of nonculturable variants.

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

Environmental fatty acids enable emergence of infectious Staphylococcus aureus resistant to FASII-targeted antimicrobials.

The bacterial pathway for fatty acid biosynthesis, FASII, is a target for development of new anti-staphylococcal drugs. This strategy is based on previous reports indicating that self-synthesized fatty acids appear to be indispensable for Staphylococcus aureus growth and virulence, although other bacteria can use exogenous fatty acids to compensate FASII inhibition. Here we report that staphylococci can become resistant to the FASII-targeted inhibitor triclosan via high frequency mutations in fabD, one of the FASII genes. The fabD mutants can be conditional for FASII and not require exogenous fatty acids for normal growth, and can use diverse fatty acid combinations (including host fatty acids) when FASII is blocked. These mutants show cross-resistance to inhibitors of other FASII enzymes and are infectious in mice. Clinical isolates bearing fabD polymorphisms also bypass FASII inhibition. We propose that fatty acid-rich environments within the host, in the presence of FASII inhibitors, might favour the emergence of staphylococcal strains displaying resistance to multiple FASII inhibitors.

A sensitive bacterial-growth-based test reveals how intestinal Bacteroides meet their porphyrin requirement.

BACKGROUND: Bacteroides sp. are dominant constituents of the human and animal intestinal microbiota require porphyrins (i.e., protoporphyrin IX or iron-charged heme) for normal growth. The highly stimulatory effect of porphyrins on Bacteroides growth lead us to propose their use as a potential determinant of bacterial colonization. However, showing a role for porphryins would require sensitive detection methods that work in complex samples such as feces. RESULTS: We devised a highly sensitive semi-quantitative porphyrin detection method (detection limit 1-4 ng heme or PPIX) that can be used to assay pure or complex biological samples, based on Bacteroides growth stimulation. The test revealed that healthy colonized or non-colonized murine and human hosts provide porphyrins in feces, which stimulate Bacteroides growth. In addition, a common microbiota constituent, Escherichia coli, is shown to be a porphyrin donor, suggesting a novel basis for intestinal bacterial interactions. CONCLUSIONS: A highly sensitive method to detect porphyrins based on bacterial growth is devised and is functional in complex biological samples. Host feces, independently of their microbiota, and E. coli, which are present in the intestine, are shown to be porphryin donors. The role of porphyrins as key bioactive molecules can now be assessed for their impact on Bacteroides and other bacterial populations in the gut.

HrtBA and menaquinones control haem homeostasis in Lactococcus lactis.

Lactococcus lactis is a fermenting Gram-positive bacterium widely used for production of dairy products. Lacking haem biosynthesis genes, L. lactis can still shift to an energetically favourable respiratory metabolism by activating a terminal cytochrome bd oxidase when haem is added to an aerated culture. Haem intracellular homeostasis is mediated by the hrtRBA operon encoding the conserved membrane HrtBA haem efflux permease and the unique intracellular haem sensor and regulator, HrtR. Here we report that membrane-associated menaquinones (MK) favour the accumulation of reduced haem in membranes. An oxidative environment, provided by oxygen, prevents and reverses haemin reduction by MK and thus limits haem accumulation in membranes. HrtBA counteracts MK-dependent membrane retention of excess haem in membrane, suggesting direct efflux from this compartment. Moreover, both HrtBA and MK-mediated reduction have a strong impact on haem intracellular pools, as determined via HrtR haem sensor induction, suggesting that intracellular haem acquisition is controlled at the membrane level without the need for dedicated import systems. Our conclusions lead to a new hypothesis of haem acquisition and regulation in which HrtBA and the bacterial membrane have central roles in L. lactis.

Following pathogen development and gene expression in a food ecosystem: the case of a Staphylococcus aureus isolate in cheese.

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.

Environmental heme utilization by heme-auxotrophic bacteria.

Heme, an iron-containing porphyrin, is the prosthetic group for numerous key cellular enzymatic and regulatory processes. Many bacteria encode the biosynthetic enzymes needed for autonomous heme production. Remarkably, however, numerous other bacteria lack a complete heme biosynthesis pathway, yet encode heme-requiring functions. For such heme-auxotrophic bacteria (HAB), heme or porphyrins must be captured from the environment. Functional studies, aided by genomic analyses, provide insight into the HAB lifestyle, how they acquire and manage heme, and the uses of heme that make it worthwhile, and sometimes necessary, to capture this bioactive molecule.

Bacterial swimmers that infiltrate and take over the biofilm matrix.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.