Swarmer Cell Development of the Bacterium Proteus mirabilisRequires the Conserved Enterobacterial Common AntigenBiosynthesis Gene rffG

Kristin Little,a Murray J. Tipping,a Karine A. Gibbsa

aDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

ABSTRACT Individual cells of the bacterium Proteus mirabilis can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarm-ing. How cells regulate this dramatic morphological remodeling remains an open ques-tion. In this paper, we move forward the understanding of this regulation bydemonstrating that P. mirabilis requires the gene rffG for swarmer cell elongation andsubsequent swarm motility. The rffG gene encodes a protein homologous to the dTDP-glucose 4,6-dehydratase protein of Escherichia coli, which contributes to enterobacterialcommon antigen biosynthesis. Here, we characterize the rffG gene in P. mirabilis, dem-onstrating that it is required for the production of large lipopolysaccharide-linked moi-eties necessary for wild-type cell envelope integrity. We show that the absence of therffG gene induces several stress response pathways, including those controlled by thetranscriptional regulators RpoS, CaiF, and RcsB. We further show that in rffG-deficientcells, the suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to inducecell elongation and swarm motility. However, the loss of RcsB does not rescue cell enve-lope integrity defects and instead results in abnormally shaped cells, including cells pro-ducing more than two poles. We conclude that an RcsB-mediated response acts to sup-press the emergence of shape defects in cell envelope-compromised cells, suggestingan additional role for RcsB in maintaining cell morphology under stress conditions. Wefurther propose that the composition of the cell envelope acts as a checkpoint beforecells initiate swarmer cell elongation and motility.

IMPORTANCE Proteus mirabilis swarm motility has been implicated in pathogenesis.We have found that cells deploy multiple uncharacterized strategies to handle cellenvelope stress beyond the Rcs phosphorelay when attempting to engage in swarmmotility. While RcsB is known to directly inhibit the master transcriptional regulatorfor swarming, we have shown an additional role for RcsB in protecting cell morphol-ogy. These data support a growing appreciation that the Rcs phosphorelay is a mul-tifunctional regulator of cell morphology in addition to its role in microbial stressresponses. These data also strengthen the paradigm that outer membrane composi-tion is a crucial checkpoint for modulating entry into swarm motility. Furthermore,the rffG-dependent moieties provide a novel attractive target for potential antimicro-bials.

KEYWORDS Proteus mirabilis, RffG, cell motility, cell surface, outer membrane, swarmdevelopment, swarm motility, swarming

Bacteria can migrate across a surface using a cooperative group motility termedswarming. For Proteus mirabilis, a Gram-negative opportunistic pathogen, rapid

surface-based swarm motility likely contributes to its pathogenesis during catheter-associated urinary tract infections (1, 2). On hard agar (1.5% to 2%) surfaces, cellselongate from �2-�m rods into hyperflagellated, snake-like “swarmer” cells that carry

Received 21 April 2018 Accepted 27 June2018

Accepted manuscript posted online 2 July2018

Citation Little K, Tipping MJ, Gibbs KA. 2018.Swarmer cell development of the bacteriumProteus mirabilis requires the conservedenterobacterial common antigen biosynthesisgene rffG. J Bacteriol 200:e00230-18. https://doi.org/10.1128/JB.00230-18.

Editor Victor J. DiRita, Michigan StateUniversity

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Karine A. Gibbs,kagibbs@mcb.harvard.edu.

RESEARCH ARTICLE

crossm

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 1Journal of Bacteriology

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

multiple chromosomes and range in length from 10 to 80 �m (3–5). Cell elongation andenhanced flagellar gene expression are considered genetically linked and occur upongrowth on a hard agar surface (6–9). Multiple swarmer cells closely associate into raftsthat collectively move across a surface (3, 5, 10). After a defined period of motility,swarmer cells divide into short (1 to 2 �m) nonmotile rod-shaped cells (11). Iterativerounds of swarmer cell elongation, group motility, and cell division comprise theswarmer cell developmental cycle and result in the rapid occupation of centimeter-scale surfaces in a stereotypical concentric ring pattern (11).

Swarmer cell elongation entails a broad range of physiological changes in additionto the dramatic morphological remodeling. Dozens of genes experience drasticchanges in expression; for example, genes for flagellum production become upregu-lated on surfaces (12–16). Elongation into swarmer cells is also coordinated withchanges to the cell envelope that minimally include alterations of the outer membrane.For example, in the outer membrane, the lipopolysaccharide (LPS) structure is modified,fluidity is increased, and areas of phospholipid bilayer arise (17–19). Cells also transitionvia unknown mechanisms from being rigid to being flexible (3–5).

In many bacteria, a bidirectional relationship exists between swarmer cell motilityand cell envelope structure. There are several cell envelope biosynthesis pathways inEnterobacteriaceae, such as LPS and enterobacterial common antigen (ECA) biosynthe-sis for the outer membrane and peptidoglycan biosynthesis for the cell wall. Interro-gating the specific contribution of each pathway to P. mirabilis swarmer cell develop-ment has proven challenging, partly because these three pathways share a pool ofsubstrates (20, 21). For example, in Escherichia coli, genetic modifications to each ofthese biosynthetic pathways can dramatically alter cell shape and motility due toperturbations in the balance of the shared cell envelope substrate, undecaprenylphosphate (20, 21). Moreover, the disruption of cell envelope-associated genes inhibitsswarmer cell development and motility of P. mirabilis through several mechanisms. Forexample, the loss of the LPS biosynthesis gene waaL (22, 23) inhibits swarmer cellelongation and motility through the activation of the Rcs phosphorelay (23), while thestress-associated sigma factor RpoE (24, 25) responds to disruptions of the LPS biosyn-thesis gene ugd (25). Less is known about the role of ECA biosynthesis in P. mirabilis.

Cell envelope structure and stress sensing also appear to play broadly conservedroles in the swarm regulation of many bacterial species, including P. mirabilis, E. coli,and Serratia marcescens (20–22, 24, 26–30). In the aforementioned organisms, the Rcs(regulator of capsule synthesis) phosphorelay, which is a complex cell envelope stress-sensing signal transduction pathway, plays a key role in swarm motility inhibition (22,26, 31). The Rcs phosphorelay, through the transcriptional regulator RcsB, directlyrepresses the flhDC genes, which themselves encode the master transcriptional regu-lator of swarming, FlhD4C2 (27, 29). The current paradigm is that cell envelope stress orouter membrane defects activate membrane-localized Rcs proteins, which then phos-phorylate and activate the response regulator RcsB (22, 26, 27, 31; see also references32 and 33 for review). Decreased levels of flhDC result in reduced flagellum productionand the failure of cells to elongate, thus inhibiting swarm motility. RcsB directlyactivates the expression of the cell division-related genes, minCDE; however, themolecular mechanisms of this regulation remain unclear (6, 7). RcsB also induces theproduction of several fimbrial genes, including paralogous genes of the fimbrialtranscriptional regulator MrpJ. Together, RcsB and MrpJ modulate broad transcriptionaland behavioral changes to promote cell adherence and biofilm formation and torepress swarm motility (7, 34).

Here, we address the role of the cell envelope and stress-sensing pathways in theregulation of swarmer cell development, an early stage of swarm motility. We show thatP. mirabilis cells require the rffG gene, which is predicted to encode the sugar-modifyingenzyme dTDP-glucose 4,6-dehydratase, to produce an uncharacterized LPS-linkedstructural component of the cell envelope. As a homologous rffG gene and its con-served cluster of flanking genes are responsible for ECA production in E. coli (35), weposit that these structures may be ECA derived. We further show that cells lacking the

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 2

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

rffG gene remain short on swarm-permissive surfaces and suffer from cell envelopeintegrity defects that make elongated cells more susceptible to rupturing. We foundthat rffG-dependent moieties were not physically required for swarmer cell elongation;instead, the loss of the rffG gene activated several swarm-inhibitory pathways, includ-ing the Rcs phosphorelay. Indeed, an RcsB-mediated response was sufficient to restrictswarmer cell elongation of rffG-deficient cells by inhibiting flhDC expression. We havealso identified a novel role for RcsB in the maintenance of cell morphology duringswarmer cell elongation. We found that RcsB was necessary to suppress cell morphol-ogy defects of rffG-deficient cells that were genetically forced to elongate into swarmercells. We posit that the cell envelope composition is a crucial signaling checkpointbefore entry into surface-based swarm motility. The Rcs phosphorelay response regu-lator not only mediates this signaling checkpoint but also serves an important role inmaintaining a normal cell shape during swarmer cell elongation.

RESULTSCells require the rffG gene to complete swarmer cell elongation and initiate

swarming. Previous research explored the role of LPS biosynthesis genes in theregulation of P. mirabilis swarm motility, but a role for ECA has not been described (23,25). Here, we interrogated the role in swarming of a gene associated with ECAbiosynthesis. We characterized a swarm-deficient mutant strain presumably incapableof producing ECA by generating a chromosomal deletion of the rffG gene in P. mirabilisstrain BB2000, resulting in a ΔrffG strain. A colony of the wild-type strain occupied astandard-size petri dish by 24 h on swarm-permissive and nutrient-rich CM55 agar;however, colonies of the ΔrffG strain did not expand beyond the site of inoculation (Fig.1A). We complemented the rffG deletion through in trans expression of the rffG geneunder the control of a lac promoter for constitutive expression in P. mirabilis (23),resulting in the ΔrffG(prffG) strain. The wild-type and the ΔrffG strain each carried emptyvectors (pBBR1-NheI) to enable growth on the same selective medium as theΔrffG(prffG) strain. The swarm colonies of the ΔrffG(prffG) strain were attenuated incomparison to those of the wild-type strain and more expansive than those of the ΔrffGstrain (Fig. 1A), indicating a partial rescue of swarm motility.

We next examined the swim motility of these strains to determine whether the lossof rffG broadly inhibits flagellum-based motility. We analyzed the motility of thewild-type, ΔrffG, and ΔrffG(prffG) strains through 0.3% LB agar, which permits swim-ming. The ΔrffG strain, and to a lesser extent the ΔrffG(prffG) strain, was delayed in theinitiation of swimming compared to the wild-type strain. However, all strains occupiedthe full petri dish within 24 h (see Fig. S1A in the supplemental material). We measuredcell viability both in liquid (Fig. S1B) and in swarms (Fig. S1C) and found that allpopulations grew to equivalent densities. Thus, the rffG gene was essential for surface-based swarm motility but not for liquid-based swimming motility or growth.

Given that cells required the rffG gene to engage in surface-based motility, wehypothesized that cells of the ΔrffG strain might fail to progress through stages ofswarming, such as increased expression of flhDC-regulated genes, elongation intoswarmer cells, or migration across a surface (Fig. 1B). Therefore, we independentlyassessed the cell morphology of the wild-type, ΔrffG, and ΔrffG(prffG) strains usingepifluorescence microscopy under swarm-permissive conditions. To visualize flagellargene expression, a Venus fluorescent protein reporter was introduced on the chromo-somes of each strain background downstream of fliA. The fliA gene encodes theflagellar sigma factor (�28) and is both directly regulated by FlhD4C2 and highlyexpressed in swarming cells (36, 37). By 4 h after inoculation onto CM55 agar at 37°C,populations of the wild-type strain contained many short nonmotile and few elongatedmotile cells (Fig. 1B). After 6 h, elongated cells expressing the fluorescent fliA reporterdominated the inoculum edge and were apparent at the leading edge of the swarms(Fig. 1B). In contrast, most cells of the ΔrffG strain were short and nonmotile at 4 and6 h; cells appeared modestly shorter than nonelongated wild-type cells (Fig. 1B). Cellsof the ΔrffG strain largely did not exhibit fliA-associated fluorescence (Fig. 1B). We

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 3

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

wildtype

pBBR1-NheI

∆rffGpBBR1-NheI

∆rffGprffG

2 hours

6 hours

4 hours

A

B

Phase

Fluorescence

Motile

DivisionElongation

Non-motile

Motile

DivisionElongation

Non-motileNon-motile

Non-motile

wildtype

pBBR1-NheI

∆rffGpBBR1-NheI

∆rffGprffG

FIG 1 Loss of the rffG gene inhibits swarmer cell elongation and swarm motility. (A) The wild-type(pBBR1-NheI), ΔrffG(pBBR1-NheI), and ΔrffG(prffG) strains were grown on swarm-permissive medium containingkanamycin as well as Coomassie blue and Congo red dyes for imaging. Images are representative of at

(Continued on next page)

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 4

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

confirmed the reduction of flagella by visualizing cells harvested from a swarm usingtransmission electron microscopy. Cells of the ΔrffG strain were uniformly short andlacked the extended structures present on the wild-type elongated swarmer cells (seeFig. S1D). The general loss of flagellar gene expression and flagellar assembly onsurfaces likely reflect differences in gene expression during liquid and surface growth,as the ΔrffG strain grown in liquid can engage in flagellum-dependent swimmingmotility (Fig. S1A). Notably, some cells of the ΔrffG strain did initiate elongation butoften ruptured or divided into short cells before completing elongation (Fig. S1E),indicating a failure to complete swarmer cell elongation. In contrast, cells of theΔrffG(prffG) strain formed elongated motile cells displaying fliA-associated fluorescenceby 6 h (Fig. 1B); this progression was delayed compared to that of the wild-type strain,which is consistent with a partial rescue. Therefore, as cells of the ΔrffG strain failed toincrease the expression of flagellar genes and to elongate into swarmer cells uponsurface contact, we concluded that the rffG gene was necessary and sufficient for cellsto initiate swarmer cell elongation.

The rffG gene is essential for the production of LPS-associated moieties nec-essary for cell envelope integrity. We considered that the lack of swarmer cellelongation in the ΔrffG strain might be caused either by a physical constraint such asa lack of membrane integrity or by activation of swarm-inhibitory signaling pathways.Therefore, we first examined the membrane composition and integrity of this strain. InP. mirabilis, ECA can exist in many forms: linked to other lipids, in a circularized andsoluble form, or surface-exposed and linked to the LPS core in the outer membrane (35,38–42). Repeated efforts to confirm the presence of ECA via Western blotting with E.coli O14 serum (SSI Diagnostica, Hillerød, Denmark), which is reactive against E.coli-derived ECA (43, 44), were unsuccessful. We instead characterized the overall LPScomposition and cell envelope sensitivity to antibiotics. We extracted LPS from surface-grown colonies of the wild-type, ΔrffG, and ΔrffG(prffG) strains and then visualized theLPS-associated moieties using silver stain (45). The observed banding patterns of thewild-type and ΔrffG(prffG) strains were nearly equivalent (Fig. 2A). The banding patternof the ΔrffG strain, however, lacked a high-molecular-weight smear, and the bandswithin the putative O-antigen ladder formed double bands instead of a single band(Fig. 2A). We concluded that the rffG gene was essential for the production of full-length and wild-type LPS, specifically, the O antigen and the high-molecular-weightcomponents.

We reasoned that these perturbations to the LPS components might cause broadercell envelope damage in cells of the ΔrffG strain. To target the outer membrane, wemeasured the resistances to polymyxin B, bile salts, and sodium dodecyl sulfate (SDS).Polymyxin B is thought to bind LPS, and the disruption of LPS biosynthesis genescauses polymyxin B sensitivity in P. mirabilis (25, 46). Bile salts (47) and SDS broadlytarget membranes through detergent-like effects. For Salmonella enterica, ECA is likelyinvolved in the resistance to bile salts (48); however, a similar role for ECA of P. mirabilishas not been explored. Populations of the wild-type and ΔrffG strains were resistant tofully saturated solutions (50 mg/ml) of polymyxin B (Table 1; see also Fig. S2A) andexhibited reduced growth on 0.5% bile salts (Table 1; Fig. S2B). However, the growthdefects of the ΔrffG strain on 0.2% bile salts were more severe than those of the

FIG 1 Legend (Continued)least three independent experiments for each strain. (B) Phase-contrast and epifluorescence microscopyof the wild-type(pBBR1-NheI), the ΔrffG(pBBR1-NheI), and the ΔrffG(prffG) strains after 2, 4, and 6 h onswarm-permissive medium containing kanamycin. All strains encode Venus immediately downstream offliA. Fluorescence corresponding to fliA reporter expression is shown for the 6-h time point. Rolling ballbackground subtraction was performed using FIJI (73). Arrowheads highlight an elongating cell in theΔrffG strain that is bulging. Frames from a time lapse of such cells bulging are in Fig. S1E in thesupplemental material. At bottom are cartoon depictions of the morphological state of cells grown onswarm-permissive solid medium. On surfaces, cells elongate up to 40-fold before engaging in motilityand dividing into short (1 to 2 �m) nonmotile cells. These morphological and behavioral changescoordinate with broad changes to the transcriptome. Images are representative of at least threeindependent experiments for each strain. Bars, 10 �m.

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 5

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

wild-type strain. The ΔrffG strain was reduced in growth and formed small andtranslucent colonies (Fig. S2B). The ΔrffG strain was also more sensitive to SDS than thewild-type strain; 0.5% SDS was permissive for the growth of the wild-type strain but notfor the ΔrffG strain (Table 1; Fig. S2C). As controls, we measured the sensitivity to thenon-membrane-targeting antibiotics gentamicin and kanamycin. We found no differ-ences in growth between the wild-type and the ΔrffG strains when grown on genta-micin and kanamycin (Table 1). In sum, the rffG-deficient cells exhibited increasedsensitivity to bile salts and SDS but not to polymyxin B, gentamicin, and kanamycin.

moieties

FIG 2 Loss of the rffG gene affects outer membrane structures and cell envelope integrity. (A) LPS was extracted from surface-grown cells of the wild-type, ΔrffG,and ΔrffG(prffG) strains. Samples were run on a 12% SDS-PAGE gel and detected via silver stain. Predicted LPS-associated moieties are labeled on the left. HMW,high molecular weight. Image is representative of at least three independent experiments for each strain. (B) The wild-type and ΔrffG strains were spread ontoswarm-permissive agar pads containing 0 or 10 �g/ml carbenicillin (Cb). Shown are cells after 1, 2, or 4 h of incubation on a surface. Images are representativeof three independent experiments. Bars,10 �m.

TABLE 1 Antibiotic sensitivity of the wild-type and ΔrffG strains on surfaces

Strain

MIC of:

Polymyxin B(mg/ml)

Bile salts(% [wt/vol])

SDS(% [wt/vol])

Gentamicin(�g/ml)

Kanamycin(�g/ml)

Wild type �50 0.2 0.05 0.1 0.1ΔrffG strain �50 0.1 0.01 0.1 0.1

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 6

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

Therefore, the outer membrane in rffG-deficient cells was compromised in a pheno-typically distinct manner than previously studied LPS-deficient P. mirabilis strains.

To further interrogate cell envelope integrity, we analyzed cell morphology inresponse to a subinhibitory concentration (10 �g/ml) of the beta-lactam antibioticcarbenicillin. Within 1 h of growth on carbenicillin-containing CM55 agar at 37°C, weobserved that wild-type cells lengthened to tens of microns while remaining uniformin diameter (Fig. 2B). By 4 h of growth, occasional bloating was apparent at midcell (Fig.2B). By contrast, after 1 h of growth on carbenicillin-containing CM55 agar at 37°C,many cells of the ΔrffG strains remained short; the few elongated cells appeared wideror lemon shaped (Fig. 2B). After 2 h of growth, the population of the ΔrffG strainconsisted of elongated bloated cells, including several with triangular protrusions (Fig.2B). By 4 h of growth, most cells of the ΔrffG strain had ruptured; the remaining cellswere several microns long (Fig. 2B). Equivalent results were attained with aztreonam, aninhibitor of the cell division protein FtsI (Fig. S2D). The rffG-deficient cells weretherefore more susceptible to cell wall stress and/or cell membrane stress andmembrane-targeting detergents, indicating that the composition of the outer mem-brane in rffG-deficient cells was compromised.

The rffG deficiency induces stress response pathways in cells. The loss of the rffG

gene resulted in altered cell envelope structure (Fig. 2A) and stability (Fig. 2B; Fig. S2D),both of which would likely induce stress response pathways. Interestingly, the rffG-deficient cells transiently elongated when artificially driven to expand in length usingcarbenicillin; therefore, the inhibited elongation in these cells was not purely due todisrupted physical structures of the cell envelope. Such cell envelope stress in P.mirabilis can activate several swarm-inhibitory pathways, such as those controlled byRcsB, RpoE, and RppA/B (22, 24, 25, 30). To identify whether one or more of thesepathways was induced in rffG-deficient cells, we performed transcriptome sequencing(RNA-Seq) analysis on cells of the wild-type and ΔrffG strains harvested under swarm-permissive conditions. Two biological replicates were acquired and examined. Shortnonmotile cells harvested from swarms of the wild-type strain were used as the controlfor these experiments.

Three hundred forty-three genes of �3,455 protein-coding genes in the P. mirabilisBB2000 genome were expressed at least 4-fold differently (see Fig. S3). One hundredthirty-six genes were decreased in the ΔrffG strain, of which approximately 18% wererelated to ribosome structure and translation and 20% were directly related to flagel-lum assembly or chemotaxis (see Table S1). Additional factors known to regulateswarmer cell development and motility also had decreased expression, including umoDat 0.12-fold, umoA at 0.19-fold, and ccm at 0.12-fold (Table 2). Cell envelope-associatedgenes were also downregulated, e.g., the penicillin-binding protein gene pbpC and themembrane lipid modifying gene ddg at 0.25-fold and 0.22-fold, respectively (Table S1).In contrast, 207 genes were increased, including the virulence-associated MR/P fimbria(200-fold-increased expression of mrpA) and Proteus P-like pili (55.5-fold-increasedexpression of pmpA) (Table 3; see Table S2). In addition, several genes related tocarnitine metabolism were increased, including the transcriptional regulator caiF at63.6-fold and caiA, fixC, and fixX at 4-fold (Tables 3 and S2). Carnitine can be metabo-lized, particularly under anaerobic conditions (49–51), and can act as a stress protectantfor several bacterial species (52, 53; also reviewed in reference 54). Likewise, ompW wasincreased �33.7-fold along with dcuB, an anaerobic C4-dicarboxylate transporter, at29.6-fold (Tables 3 and S2). In E. coli, maximal ompW expression is tied to survival in thetransition from aerobic to anaerobic growth (55). Therefore, genes for fimbrial produc-tion and for metabolism under anaerobic or microaerobic environments were morehighly expressed in the rffG-deficient cells; by contrast, genes promoting swarmmotility were decreased.

Notably, three major regulators were expressed much higher in the ΔrffG strain(Table 2): mrpJ at 19.1-fold, rpoS at 8.7-fold, and the RcsB cofactor rcsA at 9.9-fold. BothmrpJ and rcsA were previously shown to contribute to swarm inhibition (6, 7, 34). We

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 7

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

observed a partial overlap between differentially regulated genes and the characterizedMrpJ regulon (34). However, we found a larger overlap between the genes differentiallyregulated in the ΔrffG populations and the genes recently characterized as regulated byRcsB in P. mirabilis (Tables 2 and 3) (6, 7). The overlap was especially striking among thedownregulated genes. While approximately 8% of upregulated genes overlapped withthe Rcs regulon, 24% of downregulated genes overlapped with the Rcs regulon (Fig.S3). RcsB directly represses flhDC, which in turn regulates flagellar and chemotaxisgenes, positively regulates paralogous genes of the swarm-inhibitory mrpJ gene (6, 7),and regulates the minCDE genes (6). Therefore, we hypothesized that the Rcs phos-phorelay was likely activated in the rffG-deficient cells.

RcsB inhibits swarmer cell elongation and morphology defects of rffG-deficientcells. We hypothesized that RcsB-mediated repression of the swarm transcriptional

TABLE 2 Top 20 genes downregulated in the ΔrffG strain relative to the wild-type strain

Transcript(s) Product(s)Foldchange

Rcsregulona

cspB Cold shock protein 0.01flgN Flagellar synthesis protein 0.03BB2000_1016, BB2000_1017 Cold shock protein, heat shock protein 0.04flgM Anti-sigma 28 factor FlgM 0.04BB2000_3499 Lipoprotein 0.05 YesfliE Flagellar hook-basal body complex protein 0.05BB2000_1271 Putative MFS-type transporter YdeE 0.07fliA Flagellar biosynthesis sigma factor 0.08 YesBB2000_1466 Hypothetical protein YesholD DNA polymerase III subunit psi 0.10 YesintB Prophage integrase 0.11fliM, fliN Flagellar motor switch proteins FliM and FliN 0.11 YesBB2000_0342 Transcriptional regulator 0.11 YesBB2000_0996, umoD Hypothetical protein, exported protein (upregulator of flagellar master operon) 0.12 YesrpsO 30S ribosomal protein S15 0.12ccm Membrane protein (Ccm1 protein) 0.12 YesrplI 50S ribosomal protein L9 0.12fliZ Flagellar biosynthesis protein FliZ 0.12 YesBB2000_2557 Phospholipid-binding protein 0.13 YesBB2000_2289, fruK Hypothetical protein, 1-phosphofructokinase 0.13aBased on the published RcsB regulon in P. mirabilis (6, 7).

TABLE 3 Top 20 genes upregulated in the ΔrffG strain relative to the wild-type strain

Transcript ProductFoldchange

Rcsregulona

mrpA Major mannose-resistant/Proteus-like fimbrial protein 200.27 YescaiF DNA-binding transcriptional activator CaiF 63.60pmpA Fimbrial subunit 55.46BB2000_1924 Transcriptional regulator 45.63BB2000_1499 Fimbrial subunit 44.85BB2000_3017 Fimbrial operon regulator 44.59zntB Zinc transporter 42.83mrpG Fimbrial subunit 42.72BB2000_0667 Hypothetical proteinompW Outer membrane protein W 33.70BB2000_2725 TetR-family transcriptional regulator 32.64dcuB Anaerobic C4-dicarboxylate transporter 29.58BB2000_0531 Sigma 54 modulation protein 25.53BB2000_0331 Heat shock protein HtpX 22.81BB2000_0299 Hypothetical protein 22.47BB2000_0395 Hypothetical protein 21.42 YesBB2000_0229 Hypothetical protein 21.19BB2000_1497 Fimbrial chaperone protein 20.97mrpJ Fimbrial operon regulator 19.08dmsA Dimethyl sulfoxide reductase chain A 18.99 YesaBased on the published RcsB regulon in P. mirabilis (6, 7).

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 8

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

regulator genes, flhDC, was the primary cause for the loss of swarmer cell elongationand swarm motility of the ΔrffG strain. Therefore, we independently constructed achromosomal deletion of the rcsB gene in the wild-type and the ΔrffG strains, resultingin the ΔrcsB and ΔrffG ΔrcsB strains, respectively. We also constructed a plasmid forconstitutive and increased expression of flhDC (under the lac promoter) and introducedthis in trans in the wild-type and the ΔrffG strains, resulting in the wild-type(pflhDC)strain and the ΔrffG(pflhDC) strain, respectively. As RcsB is an upstream repressor offlhDC, we predicted that swarm motility and swarmer cell elongation would be rescuedin both the ΔrffG ΔrcsB and the ΔrffG(pflhDC) strains. The wild-type(pflhDC) strain andthe ΔrcsB strain were predicted to contain constitutively swarming cells on the basis ofequivalent constructs in other P. mirabilis wild-type backgrounds (9, 29). We inoculatedall strains on separate swarm-permissive agar plates and analyzed colony expansionover 16 h of growth at 37°C (Fig. 3). As predicted, the wild-type(pflhDC) strain and theΔrcsB strain expanded across the petri dish (swarm radii of 45 � 0 mm) by 16 h (Fig.3). However, neither the ΔrffG ΔrcsB nor ΔrffG(pflhDC) strain fully occupied the plate by16 h (Fig. 3A). The ΔrffG(pflhDC) strain did reach the edge of the plate by 24 h (swarmradius of 45 � 0 mm) (see Fig. S4), but the ΔrffG ΔrcsB strain remained constrainedtoward the center, with a swarm radius of 20.1 mm � standard deviation of 3.4 mm (seeFig. S4). Extracted LPSs of these strains grown in liquid broth and on surfaces wereanalyzed. We found that the banding pattern of the ΔrcsB strain was equivalent to thatof the wild-type strain (see Fig. S5). Likewise, the banding patterns of the ΔrffG ΔrcsBand the ΔrffG(pflhDC) strains were equivalent to that of the ΔrffG strain (Fig. S5). Thus,neither RcsB nor FlhD4C2 contributed to the production of the rffG-dependent LPS-associated moieties. However, increased expression of flhDC or deletion of rcsB wassufficient to increase swarm motility of rffG-deficient cells, indicating that RcsB-mediated repression of flhDC was sufficient for the swarm inhibition of rffG-deficientcells.

Since the ΔrffG ΔrcsB strain did not have full recovery of swarm motility by 24 h, wehypothesized that the loss of the rcsB gene might affect a pathway separate from thatof the FlhD4C2-regulated genes. We integrated the chromosomal fliA-Venus transcrip-tional reporter into each strain and observed the resultant cells using epifluorescencemicroscopy under swarm-permissive conditions. After 6 h of growth at 37°C, thewild-type(pflhDC)-derived and the ΔrffG(pflhDC)-derived strains consisted of motileelongated cells with fliA reporter-associated fluorescence (Fig. 4). Likewise, cells in theΔrcsB strain-derived strain were generally elongated and motile with fliA reporter-associated fluorescence (Fig. 4). Surprisingly, cells of the ΔrffG ΔrcsB strain-derivedstrain exhibited severe cell shape defects: cells were bloated and uneven in width,

2 4 6 8 10 12 14 16

0

10

20

30

40

50

∆rffG pflhDCwildtype pflhDC

∆rcsB pBBR1-NheI

∆rffG∆rcsB pBBR1-NheI

Sw

arm

rad

ius

(mm

)

Hours

FIG 3 Loss of the rffG gene impacts swarm colony development. Loss of the rffG gene extended thelag phase before swarm colony expansion. Liquid-grown populations of the wild-type(pflhDC), theΔrffG(pflhDC), the ΔrcsB(pBBR1-NheI), and the ΔrffG ΔrcsB(pBBR1-NheI) strains were normalized onthe basis of the OD600 and inoculated onto swarm-permissive plates containing kanamycin for plasmidretention. Swarm radii of visible swarm colony expansions were measured at indicated times; each timepoint comprises separate plates. Three independent experiments were performed for each strain andtime point.

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 9

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

forming spheres, tapering at the cell poles, or bulging at midcell (Fig. 4). In addition,several cells of the ΔrffG ΔrcsB strain were forked at the cell pole or branched at midcell,resulting in the formation of more than two cell poles. Similar shape defects were notobserved in cells of the ΔrcsB strain-derived population nor have they been reported forpreviously described P. mirabilis (6, 29) or E. coli (20, 56) ΔrcsB mutant strains. None-theless, the elongated cells of the ΔrffG ΔrcsB strain-derived strain exhibited fliAreporter-associated fluorescence and were motile (Fig. 4). In sum, cells of the ΔrffGΔrcsB strain-derived strain did not retain fidelity of a two-pole, rod-shaped swarmer cellmorphology, even though they had increased fliA expression. Thus, RcsB contributed tothe suppression of shape defects in rffG-deficient cells. As these defects only arose inthe absence of RcsB, we posit this was achieved via an RcsB-dependent and FlhD4C2-independent pathway(s).

DISCUSSION

Here crucial insights were elucidated about the role of cell envelope structure andstress sensing in the development of P. mirabilis swarmer cells, specifically regardingthe cell envelope biosynthesis gene rffG and the signaling pathways that respond to itsabsence (Fig. 5). We have shown that the rffG gene was essential for the assembly ofa swarm-permissive cell envelope. The loss of the rffG gene resulted in the loss ofLPS-associated moieties, the alteration of the O-antigen ladder, and increased sensitiv-ity to antimicrobials that specifically target the cell envelope. On the basis of theRNA-Seq results, cells of the ΔrffG strain entered into a distinctive transcriptional state,

eydenarbmeMesahP fliA )nimooz(enarbmeMretroper

∆rcsB

wildtype

pflhDC

∆rffGpflhDC

∆rffG∆rcsB

FIG 4 Swarm cell elongation of the ΔrffG strain is rescued by increased flhDC expression or deletion ofthe rcsB gene. Epifluorescence microscopy of the wild-type(pflhDC), the ΔrffG(pflhDC), the ΔrcsB, and theΔrffG ΔrcsB strains on swarm-permissive agar pads. All strains encode Venus controlled by the promoterfor fliA. For populations of the wild-type(pflhDC) strain and the ΔrffG(pflhDC) strain, the agar containedkanamycin for plasmid retention. Images were taken once swarm motility initiated (4 to 6 h on surface).Rolling ball background subtraction on fliA reporter images was conducted using FIJI (63). Arrowheadsindicate a cell exhibiting shape and polarity defects. Cropped images are shown on the far right foremphasis on cell shape defects. Left, phase contrast; middle left, Venus expression; middle right,membrane stain; far right, cropped selection of membrane stain image at higher zoom. Images arerepresentative of at least three independent experiments for each strain. Bars, 10 �m.

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 10

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

resulting in the upregulation of several stress response pathways. While most of thepathways activated by the loss of rffG have yet to be characterized, RcsB-mediatedinhibition of flhDC expression was a major regulatory factor in restricting elongation inthe ΔrffG strain. The loss of rcsB or overexpression of flhDC rescued swarm motility inthe ΔrffG strain. Moreover, an additional role for RcsB in the maintenance of cell shapeand polarity during swarmer cell elongation was uncovered, as RcsB served to alsomaintain the two-pole rod shape of rffG-deficient cells.

The transcriptional state of cells in the ΔrffG strain was characterized by theactivation of pathways such as those controlled by the transcriptional regulators RpoS,CaiF, and RcsB. There was increased expression of several fimbrial gene clusters. Therewas also a notable increase in carnitine metabolism genes, which is associated withgrowth under anaerobic conditions (50, 51). Many of the identified genes are controlledby MrpJ and oxygen availability (57), raising the possibility that cells of the ΔrffG strainbias toward a more adherent low-oxygen lifestyle. Flagellar and chemotaxis genes haddecreased expression in the ΔrffG strain. The disruption of the flagellar pathway wasconsistent with the loss of swarm motility in the ΔrffG strain. However, the potentialmechanisms for inhibiting cell elongation and driving cell shape defects were lessapparent in the RNA-Seq data. For example, while RcsB has been implicated in cellelongation via the regulation of minCDE (6, 7), differential regulation of these genes inthe ΔrffG strain was not evident. Further research will need to be done to completelycategorize the genes differentially regulated in rffG-deficient cells and to fully under-stand the physiological and behavioral implications of these altered expression levels.

Several questions remain regarding the mechanisms of activation, as well as thedownstream activity, of RcsB and MrpJ in the ΔrffG strain. First, Bode et al. recentlydemonstrated that MrpJ acts as a regulator mediating the transition of cells betweenswarm motility (MrpJ repressed) and nonmotile adherence (MrpJ induced) similar toRcsB (34). MrpJ and RcsB may positively regulate each other and have overlappingregulons (6, 7, 34), making it difficult to genetically disentangle the contributions ofeach regulator. Additionally, the perturbation of outer membrane structures appears tobe communicated to the Rcs phosphorelay through both RcsF-dependent and-independent pathways in P. mirabilis (22). Whether cell envelope stress of rffG-deficientcells is communicated through the outer membrane-localized RcsF, through the up-regulation of RcsA, or through an uncharacterized additional pathway remains to be

Activated Rcs

FlhD4C

2

RcsB

flhDC

Cell shape Swarmer cell elongation

and motility

rffG deficiency

(outer membrane

damage)

Additional

responses,

e.g., CaiF,

RpoS

Fimbriae, aerobic

metabolism, and

other pathways

FIG 5 Checkpoint model in which rffG deficiency induces multiple stress-associated pathways controllingswarmer cell elongation and cell shape. Loss of the rffG gene induced activity of RcsB, which in turndirectly repressed the expression of the flhDC genes that encode the master regulator of flagellumproduction and swarm motility. We found that RcsB in P. mirabilis was also necessary to maintain cellshape and polarity through yet uncharacterized mechanisms, supporting a role for RcsB as a multifunc-tional regulator of swarmer cell development and motility. The composition of the cell envelope,including the rffG-dependent moieties, may serve as a developmental checkpoint before engaging inswarmer cell development. Cell elongation and increased flhDC expression are initial steps in swarmercell development.

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 11

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

determined. Also unknown is whether the rffG-dependent LPS-associated moietiescommunicate to Rcs via the Umo system, as was previously shown for O antigen (22).

Previous research has elucidated how disrupting LPS induces stress response path-ways and leads to swarm inhibition. For example, abrogation of the O-antigen structurethrough the deletion of the O-antigen ligase (waaL) or chain length determinant (wzz)inhibits the activation of flhDC upon surface contact (23). The loss of the sugar-modifying O-antigen biosynthesis genes ugd and galU inhibits swarmer cell elongationand motility (25). The aforementioned genes are implicated in LPS biosynthesis. Here,we propose that P. mirabilis cells require cell envelope structures in addition to LPS forthe initiation of swarmer cell elongation (Fig. 5). The rffG-dependent high-molecular-weight LPS-associated moieties are not chemically characterized; we hypothesize thatthese might consist of LPS-associated ECA or ECA-derived moieties, since the E. coli rffGhomologue is needed for the production of the broadly conserved ECA (35). Furtherresearch is needed to characterize the structural changes to the outer membrane inrffG-deficient cells, especially as these moieties contribute to the overall membraneintegrity on surfaces.

The outer membrane structure also plays a crucial mechanical role in resisting turgorpressure fluctuations associated with cell wall stress, specifically, by beta-lactam drugs(58). Cells of the ΔrffG strain were sensitive to detergent-like membrane-targetingantimicrobials, altogether suggesting that the rffG-dependent moieties are crucial forthe outer membrane composition and integrity. One explanation is that these rffG-associated cell envelope defects are caused by pleiotropic effects resulting fromdisrupting a cell envelope biosynthesis pathway that uses a shared pool of precursormolecules. We raise this possibility because the perturbation of ECA or LPS biosynthesisgenes in E. coli causes the accumulation of dead-end intermediates that broadly impactcell envelope integrity (20, 21). However, we instead posit that the cell envelope defectsin rffG-deficient cells might be sufficient to sensitize the cells to form defective cellshapes.

We propose that RcsB acts to suppress cell wall defects in rffG-deficient cells as wellas potentially other cell-envelope compromised cells (Fig. 5). We observed that theabsence of the rffG-dependent moieties did not mechanically restrict swarmer cellelongation or result in the formation of more than two poles in artificially elongatedcells constitutively expressing flhDC from a nonnative promoter. Indeed, the popula-tions of the ΔrffG(pflhDC) strain occupied a standard-sized petri dish in 24 h, thoughthese populations exhibited a lower rate of swarm colony expansion than populationsof the wild-type(pflhDC) strain. These results suggest that rffG-mediated cell defectsmay impact swarm motility independent of flhDC regulation. The cells lacking both rffGand rcsB exhibited growth from the midcell and the formation of more two cell polesin addition to other physical perturbations. Thus, although the deletion of rcsB rescuescell elongation and motility through the derepression of flhDC, the absence of RcsB alsoperturbed a yet uncharacterized morphology-generating pathway critical for the cellshape and integrity of rffG-deficient cells. Others have also proposed that the Rcsphosphorelay plays a conditional role in cell shape maintenance in other bacteria.L-form E. coli cells require the Rcs phosphorelay to recover a rod shape; cells lacking thisresponse rupture (59). The authors of that study proposed that Rcs might function tomaintain cell shape under conditions in which cells lose the cell wall through exposureto lysozyme or cationic antimicrobial peptides, including in several niches within ahuman host (59). Moreover, in E. coli and Agrobacterium tumefaciens, cell polaritydefects similar to those of the ΔrffG ΔrcsB strain appear to arise from the formation ofpatches of inert peptidoglycan and mislocalized division planes (60–64). Further studyis needed to mechanistically understand which aspects of the RcsB regulon are specificfor cell shape maintenance and how additional poles emerge in cells lacking both RcsBand the rffG-dependent moieties.

It remains unclear how RcsB, which is presumably inactive in swarming cells, canplay a role in swarmer cell shape maintenance. We propose two broad mechanisms thatmay resolve this contradiction. First, the role(s) of RcsB in cell shape maintenance may

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 12

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

occur prior to the initiation of swarmer cell elongation. Elongation may exacerbateunrepaired envelope flaws that manifest in cell shape and polarity defects. As such, theRcs phosphorelay would act as a developmental checkpoint to restrict swarmer celldevelopment under conditions challenging to the cell envelope. Second, RcsB mayhave multiple states beyond simply “active” and “inactive” that may enable differentialactivity across time and cell states. The DNA-binding activity of RcsB has been shownto be modulated by both phosphorylation and the association with auxiliary transcrip-tion factors in E. coli (65). How RcsB activity is modulated downstream of phosphory-lation and the association with potential auxiliary transcription factors remain unknownin P. mirabilis.

Altogether, we propose that cell envelope stress, such as the absence of rffG-dependent moieties, functions as a developmental checkpoint before swarmer cellelongation and increased flagellar gene expression (Fig. 5). Under swarm-permissiveconditions in the presence of a wild-type cell envelope structure, the Rcs phosphorelay,along with other stress-sensing pathways, are inactive, thereby enabling the swarmerdevelopment to progress. When the cell envelope is perturbed, we posit that theactivity of cell envelope stress response sensors culminates in the adaptation of anadherence-promoting lifestyle that may provide protection against external stressors.As the Rcs phosphorelay, flhDC, and rffG, among other discussed genes, are conservedamong the Enterobacteriaceae family, we predict these factors may broadly serve tomodulate bacterial swarm motility and potentially cell development.

MATERIALS AND METHODSGrowth conditions. Liquid cultures were grown in Lennox broth (LB). Colonies were grown in 0.3% LB

agar for swimming motility assays, on low-swarm (LSW�) agar (66) for plating nonmotile colonies, and onCM55 blood agar base (Oxoid, Hampshire, UK) for swarming. The antibiotics for selection used throughout allassays were as follows: 15 �g/ml tetracycline (Amresco Biochemicals, Solon, OH), 25 �g/ml streptomycin(Sigma-Aldrich, St. Louis, MO), and 35 �g/ml kanamycin (Corning, Corning, NY). For swarm assays, overnightcultures were normalized to an optical density at 600 nm (OD600) of 1.0, and 1 �l of culture was inoculatedwith a needle onto swarm-permissive CM55 blood agar base (Oxoid, Hampshire, UK) plates containing 40�g/ml Congo red, 20 �g/ml Coomassie blue, and kanamycin (Corning, Corning, NY) as needed. Plates wereincubated at 37°C. When indicated, we used strains carrying an empty vector (pBBR1-NheI [67]) to conferkanamycin resistance. Images were taken with a Canon EOS 60D camera.

Strain construction. Strain construction was performed as described previously (68). The strains andplasmids are listed in Table 4. All plasmids were confirmed by Sanger sequencing (Genewiz, SouthPlainfield, NJ). For all strains, expression plasmids were introduced into P. mirabilis via E. coli SM10�pir aspreviously described (66). The resultant strains were confirmed by PCR of the targeted region. The ΔrffGstrain was additionally confirmed through whole-genome sequencing as described in reference 69.

For the construction of the ΔrcsB strain, a gBlock (Integrated DNA Technologies, Coralville, IA)containing the 452 bp upstream and downstream of rcsB (P. mirabilis BB2000, accession numberCP004022; nucleotides [nt] 1972701 to 1973153 and 1973809 to 1974261) was generated and introducedto pKNG101 at SpeI and XmaI sites using SLiCE (70). Similarly, for the construction of the ΔrffG strain, agBlock containing a chloramphenicol resistance cassette (amplified from pBAD33 [71]) flanked by 1,000bp upstream of rffG and downstream of rffG (P. mirabilis BB2000, accession number CP004022; nt3635400 to 3636399 and 3637473 to 3638473) (Integrated DNA Technologies, Coralville, IA) wasintroduced to pKNG101 (68) at the same sites. For the construction of fliA reporter strains, a gBlockencoding the last 500 bp of fliA (P. mirabilis BB2000, accession number CP004022; nt 1856328 to1856828), ribosomal binding site (RBS; AGGAGG), a modified variant of Venus fluorescent protein (a giftfrom Enrique Balleza and Philippe Cluzel [72]), and 500 bp downstream of fliA (P. mirabilis BB2000,accession number CP004022; nt 1855828 to 1856328) (Integrated DNA Technologies, Coralville, IA) wasinserted into pKNG101 (68) at the ApaI and XbaI sites. For the construction of the rffG expression strains,the nucleotide sequence for rffG was amplified via PCR from the P. mirabilis chromosome using oKL273and oKL274 and inserted into expression vector pBBR1-NheI (67) using AgeI and NheI restriction enzymesites. For the flhDC expression strains, the nucleotide sequence of flhDC (amplified using oKL277 andoKL278) was similarly inserted into pBBR1-NheI. A gBlock containing the lac promoter (Integrated DNATechnologies, Coralville, IA) was introduced upstream of the coding region using SLiCE (70).

LPS extraction and analysis. Cells were grown overnight at 37°C on swarm-permissive CM55 (Oxoid,Hampshire, UK) agar with antibiotics as needed and harvested with LB. LPS from cells was extracted usingan LPS extraction kit according to the manufacturer’s instructions (iNtRON Biotechnology Inc., Seong-nam, Gyeonggi, South Korea). Extracts were resuspended in 10 mM Tris buffer (pH 8.0) and run on a 12%SDS-PAGE gel. The gels were stained with a modified silver stain protocol (45).

Halo assays (MIC determination). Cultures were top-spread on LSW� medium and allowed to sit onthe benchtop until the surface appeared dry (a couple of hours). Six-millimeter sterile filter disks wereplaced on the plates and soaked with 10 �l of dilutions containing polymyxin B (Sigma-Aldrich, St. Louis,MO), gentamicin (Calbiochem, San Diego, CA), or kanamycin (Corning, Corning, NY) in water. A water-

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 13

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

alone control was included. Once the filter disks dried (a couple of hours), the plates were incubated at37°C overnight and imaged.

Resistance assays to bile salts and sodium dodecyl sulfate. Cultures were grown overnight,normalized to an OD of 1.0, and serially diluted 10-fold. One-microliter spots of 10�1 to 10�8 dilutionsof each strain (in technical triplicates) were inoculated onto LSW� plates containing bile salts (Sigma-Aldrich, St. Louis, MO) or sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO) (filter sterile, added tomedium after autoclaving). The plates were incubated overnight at 37°C.

Carbenicillin sensitivity assay. Cells were harvested from swarm-permissive medium using LB andspread onto 1-mm CM55 (Oxoid, Hampshire, UK) agar pads containing 10 �g/ml carbenicillin (Corning,Corning, NY). The cells were imaged after 1, 2, or 4 h on the surface. Three biological replicates wereanalyzed.

Microscopy. Microscopy was performed as previously described (69). Briefly, CM55 (Oxoid, Hamp-shire, UK) agar pads, supplemented as needed with 35 �g/ml kanamycin for plasmid retention, wereinoculated from overnight stationary cultures and incubated at 37°C in a modified humidity chamber.The pads were imaged using a Leica DM5500B (Leica Microsystems, Buffalo Grove, IL) and a CoolSnapHQ2 cooled charge-coupled-device (CCD) camera (Photometrics, Tucson, AZ). MetaMorph version 7.8.0.0(Molecular Devices, Sunnyvale, CA) was used for image acquisition. The images were analyzed using FIJI(73) (National Institutes of Health, USA); where indicated, the images were subjected to backgroundsubtraction equally across the entire image. Where indicated, the cells were stained with 25 �M1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH; Invitrogen,Carlsbad, CA) (maximum excitation, 355 nm; maximum emission, 430 nm) imaged in the DAPI (4=,6-diamidino-2-phenylindole) channel using an A4 filter cube (excitation, 360/40 nm; emission, 470/40 nm)(Leica Microsystems, Buffalo Grove, IL). Venus (maximum excitation, 515 nm; maximum emission, 528nm) was visualized in the green fluorescent protein (GFP) channel using a GFP ET filter cube (excitation,470/40 nm; emission, 525/50 nm [Leica Microsystems, Buffalo Grove, IL]). The fluorescence intensity andexposure time for each fluorescence channel were equivalent across all fluorescence microscopyexperiments. Fluorescence due to Venus was not quantified and was visible due to the overlappingexcitation and emission spectra with the GFP ET filter cube.

Transcriptional analysis. Strains were grown on CM55 (Oxoid, Hampshire, UK) plates at 37°C. Forwild-type samples, the colonies were inoculated on CM55 (Oxoid, Hampshire) agar and incubatedovernight for swarm development. The presence of short nonmotile cells in consolidation phase wasconfirmed by light microscopy. Wild-type cells from the swarm edge were then harvested by scrapingwith a plastic loop into 1 ml of RNA Protect solution (Qiagen, Venlo, Netherlands). Samples of the ΔrffGstrain were harvested after an overnight incubation by scraping whole colonies into 1 ml RNA Protect

TABLE 4 Strains and plasmids used in this study

Strain or plasmid Description Source or reference

StrainsBB2000 P. mirabilis wild type 65KEL01 BB2000 rffG::Cmr This studyKEL01(prffG) ΔrffG strain carrying plasmid prffG, which encodes constitutive rffG

expressionThis study

BB2000(pflhDC) BB2000 carrying plasmid pflhDC, which encodes constitutive flhDC expression This studyKEL01(pflhDC) ΔrffG strain carrying plasmid pflhDC, which encodes constitutive flhDC

expressionThis study

KEL02 BB2000 ΔrcsB This studyKEL03 BB2000 rffG::Cmr ΔrcsB This studyBB2000 fliA-venus BB2000 with an RBSa and the sequence encoding Venus inserted

immediately downstream of the fliA stop codonThis study; Venus construct from

reference 71BB2000 fliA-venus(pflhDC) BB2000 carrying plasmid pflhDC with an RBS and the sequence encoding

Venus inserted immediately downstream of the fliA stop codonThis study

KEL01 fliA-venus ΔrffG strain with an RBS and the sequence encoding Venus insertedimmediately downstream of the fliA stop codon

This study; Venus construct fromreference 71

KEL01 fliA-venus(pflhDC) ΔrffG strain carrying plasmid pflhDC with an RBS and the sequence encodingVenus inserted immediately downstream of the fliA stop codon

This study

KEL02 fliA-venus ΔrcsB strain with an RBS and the sequence encoding Venus insertedimmediately downstream of the fliA stop codon

This study; Venus construct fromreference 71

KEL03 fliA-venus ΔrffG ΔrcsB strain with an RBS and the sequence encoding Venus insertedimmediately downstream of the fliA stop codon

This study; Venus construct fromreference 71

PlasmidspBBR1-NheI Empty vector 66prffG pBBR1-NheI backbone containing the rffG gene under the lac promoter,

resulting in constitutive expression in P. mirabilisThis study

pflhDC pBBR1-NheI backbone containing the flhDC genes under the lac promoter,resulting in constitutive expression in P. mirabilis

This study

aRBS, ribosomal binding site.

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 14

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

solution. Total RNA was isolated using an RNeasy minikit (Qiagen, Venlo, Netherlands) according to themanufacturer’s instructions. RNA purity was measured using an Agilent 2200 Tapestation (Agilent, SantaClara, CA). To enrich mRNA, rRNA was digested using terminator 5= phosphate-dependent exonuclease(Illumina, San Diego, CA) according to the manufacturer’s instructions and purified by phenol-chloroformextraction (74). cDNA libraries were prepared from mRNA-enriched RNA samples using an NEBNext UltraRNA library preparation kit (New England BioLabs, Ipswich, MA) according to the manufacturer’sinstructions. The libraries were sequenced on an Illumina NextSeq 2500 instrument with 250-base pairsingle-end reads at the Harvard University Bauer Core. Sequences were matched to the BB2000 referencegenome (accession number CP004022) using TopHat 2 (75). Differential expression data were generatedusing the Cufflinks RNA-Seq analysis suite (76) run on the Harvard Odyssey cluster, courtesy of theHarvard University Research Computing Group. The data were analyzed using the CummeRbundpackage for R and Microsoft Excel (76). Bioinformatics information was derived from KEGG (77). The datain this paper represent the combined analyses of two independent biological repeats.

Accession number(s). Data for this study have been deposited in the GEO database under accessionno. GSE116587. Accession numbers for individual data sets are GSM3243501, GSM2343502, GSM3243503,and GSM3243504.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00230-18.

SUPPLEMENTAL FILE 1, PDF file, 8.5 MB.

ACKNOWLEDGMENTSWe thank Enrique Balleza and Philippe Cluzel for the kind gift of the Venus construct

and members of the Gibbs, D’Souza, Gaudet, and Losick labs for thoughtful discussionand feedback. Doug Richardson, Sven Terclavers, and Sebastian Gliem assisted withimaging on the Cell Observer. We thank the staff of the Bauer Core at HarvardUniversity for assistance with RNA-Seq and Maria Ericsson of the Harvard MedicalSchool Electron Microscopy facility for assistance with transmission electron micros-copy (supplemental material).

This research was funded by a Smith Family Graduate Fellowship in Science andEngineering, a Simmons Family Award for the Harvard Center for Biological Imaging, theDavid and Lucile Packard Foundation, The George W. Merck Fund, and Harvard University.

We declare no conflicts of interest.K.L. and K.A.G. conceived and coordinated the study and wrote the paper. K.L.

performed all experiments, except the RNA-Seq, which was performed by M.J.T. Allauthors edited the paper.

REFERENCES1. Armbruster CE, Forsyth-DeOrnellas V, Johnson AO, Smith SN, Zhao L, Wu W,

Mobley HLT. 2017. Genome-wide transposon mutagenesis of Proteusmirabilis: essential genes, fitness factors for catheter-associated urinary tractinfection, and the impact of polymicrobial infection on fitness requirements.PLoS Pathog 13:e1006434. https://doi.org/10.1371/journal.ppat.1006434.

2. Burall LS, Harro JM, Li X, Lockatell CV, Himpsl SD, Hebel JR, Johnson DE,Mobley HLT. 2004. Proteus mirabilis genes that contribute to pathogen-esis of urinary tract infection: identification of 25 signature-taggedmutants attenuated at least 100-fold. Infect Immun 72:2922–2938.https://doi.org/10.1128/IAI.72.5.2922-2938.2004.

3. Hoeniger J. 1965. Development of flagella by Proteus mirabilis. Microbi-ology 40:29 – 42.

4. Hoeniger JF. 1966. Cellular changes accompanying the swarming ofProteus mirabilis. II. Observations of stained organisms. Can J Microbiol12:113–123.

5. Hauser G. 1885. Ueber fäulnissbacterien und deren beziehungen zursepticämie: ein beitrag zur morphologie der spaltpilze. Vogel, Leipzig,Germany.

6. Howery KE, Clemmer KM, Simsek E, Kim M, Rather PN. 2015. Regulationof the Min cell division inhibition complex by the Rcs Phosphorelay inProteus mirabilis. J Bacteriol 197:2499 –2507. https://doi.org/10.1128/JB.00094-15.

7. Howery KE, Clemmer KM, Rather PN. 2016. The Rcs regulon in Proteusmirabilis: implications for motility, biofilm formation, and virulence. CurrGenet 62:775–789. https://doi.org/10.1007/s00294-016-0579-1.

8. Prüss BM, Matsumura P. 1996. A regulator of the flagellar regulon ofEscherichia coli, flhD, also affects cell division. J Bacteriol 178:668 – 674.

9. Furness RB, Fraser GM, Hay NA, Hughes C. 1997. Negative feedback froma Proteus class II flagellum export defect to the flhDC master operoncontrolling cell division and flagellum assembly. J Bacteriol 179:5585–5588. https://doi.org/10.1128/jb.179.17.5585-5588.1997.

10. Jones BV, Young R, Mahenthiralingam E, Stickler DJ. 2004. Ultrastructureof Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection. Infect Immun 72:3941–3950. https://doi.org/10.1128/IAI.72.7.3941-3950.2004.

11. Rauprich O, Matsushita M, Weijer CJ, Siegert F, Esipov SE, Shapiro JA.1996. Periodic phenomena in Proteus mirabilis swarm colony develop-ment. J Bacteriol 178:6525– 6538. https://doi.org/10.1128/jb.178.22.6525-6538.1996.

12. Pearson MM, Rasko DA, Smith SN, Mobley HLT. 2010. Transcriptome ofswarming Proteus mirabilis. Infect Immun 78:2834 –2845. https://doi.org/10.1128/IAI.01222-09.

13. Allison C, Lai HC, Hughes C. 1992. Co-ordinate expression of virulencegenes during swarm-cell differentiation and population migration ofProteus mirabilis. Mol Microbiol 6:1583–1591. https://doi.org/10.1111/j.1365-2958.1992.tb00883.x.

14. Clemmer KM, Rather PN. 2008. The Lon protease regulates swarmingmotility and virulence gene expression in Proteus mirabilis. J Med Mi-crobiol 57:931–937. https://doi.org/10.1099/jmm.0.47778-0.

15. Fraser GM, Claret L, Furness R, Gupta S, Hughes C. 2002. Swarming-

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 15

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

coupled expression of the Proteus mirabilis hpmBA haemolysin operon.Microbiology 148:2191–2201. https://doi.org/10.1099/00221287-148-7-2191.

16. Morgenstein RM, Szostek B, Rather PN. 2010. Regulation of geneexpression during swarmer cell differentiation in Proteus mirabilis.FEMS Microbiol Rev 34:753–763. https://doi.org/10.1111/j.1574-6976.2010.00229.x.

17. Armitage JP, Smith DG, Rowbury RJ. 1979. Alterations in the cell enve-lope composition of Proteus mirabilis during the development ofswarmer cells. Biochim Biophys Acta 584:389 –397. https://doi.org/10.1016/0304-4165(79)90115-6.

18. Armitage JP. 1982. Changes in the organization of the outer membraneof Proteus mirabilis during swarming: freeze-fracture structure and mem-brane fluidity analysis. J Bacteriol 150:900 –904.

19. Gué M, Dupont V, Dufour A, Sire O. 2001. Bacterial swarming: a bio-chemical time-resolved FTIR-ATR study of Proteus mirabilis swarm-celldifferentiation. Biochemistry 40:11938 –11945. https://doi.org/10.1021/bi010434m.

20. Jorgenson MA, Kannan S, Laubacher ME, Young KD. 2016. Dead-endintermediates in the enterobacterial common antigen pathway inducemorphological defects in Escherichia coli by competing for undecaprenylphosphate. Mol Microbiol 100:1–14. https://doi.org/10.1111/mmi.13284.

21. Jorgenson MA, Young KD. 2016. Interrupting biosynthesis of O antigenor the lipopolysaccharide core produces morphological defects in Esch-erichia coli by sequestering undecaprenyl phosphate. J Bacteriol 198:3070 –3079. https://doi.org/10.1128/JB.00550-16.

22. Morgenstein RM, Rather PN. 2012. Role of the Umo proteins and the Rcsphosphorelay in the swarming motility of the wild type and anO-antigen (waaL) mutant of Proteus mirabilis. J Bacteriol 194:669 – 676.https://doi.org/10.1128/JB.06047-11.

23. Morgenstein RM, Clemmer KM, Rather PN. 2010. Loss of the waaLO-antigen ligase prevents surface activation of the flagellar gene cas-cade in Proteus mirabilis. J Bacteriol 192:3213–3221. https://doi.org/10.1128/JB.00196-10.

24. Liu M-C, Kuo K-T, Chien H-F, Tsai Y-L, Liaw S-J. 2015. New aspects of RpoEin uropathogenic Proteus mirabilis. Infect Immun 83:966 –977. https://doi.org/10.1128/IAI.02232-14.

25. Jiang SS, Lin TY, Wang WB, Liu MC, Hsueh PR, Liaw SJ. 2010. Character-ization of UDP-glucose dehydrogenase and UDP-glucose pyrophospho-rylase mutants of Proteus mirabilis: defectiveness in polymyxin B resis-tance, swarming, and virulence. Antimicrob Agents Chemother 54:2000 –2009. https://doi.org/10.1128/AAC.01384-09.

26. Castelli ME, Véscovi EG. 2011. The Rcs signal transduction pathway istriggered by enterobacterial common antigen structure alterations inSerratia marcescens. J Bacteriol 193:63–74. https://doi.org/10.1128/JB.00839-10.

27. Francez-Charlot A, Laugel B, Van Gemert A, Dubarry N, Wiorowski F,Castanié-Cornet M-P, Gutierrez C, Cam K. 2003. RcsCDB His-Asp phos-phorelay system negatively regulates the flhDC operon in Escherichiacoli. Mol Microbiol 49:823– 832. https://doi.org/10.1046/j.1365-2958.2003.03601.x.

28. Toguchi A, Siano M, Burkart M, Harshey RM. 2000. Genetics of swarmingmotility in Salmonella enterica serovar Typhimurium: critical role forlipopolysaccharide. J Bacteriol 182:6308 – 6321. https://doi.org/10.1128/JB.182.22.6308-6321.2000.

29. Clemmer KM, Rather PN. 2007. Regulation of flhDC expression in Proteusmirabilis. Res Microbiol 158:295–302. https://doi.org/10.1016/j.resmic.2006.11.010.

30. Wang WB, Chen I-C, Jiang SS, Chen HR, Hsu CY, Hsueh PR, Hsu WB, LiawSJ. 2008. Role of RppA in the regulation of polymyxin B susceptibility,swarming, and virulence factor expression in Proteus mirabilis. InfectImmun 76:2051–2062. https://doi.org/10.1128/IAI.01557-07.

31. Laubacher ME, Ades SE. 2008. The Rcs phosphorelay is a cell envelopestress response activated by peptidoglycan stress and contributes tointrinsic antibiotic resistance. J Bacteriol 190:2065–2074. https://doi.org/10.1128/JB.01740-07.

32. Majdalani N, Gottesman S. 2005. The Rcs phosphorelay: a complex signaltransduction system. Annu Rev Microbiol 59:379 – 405. https://doi.org/10.1146/annurev.micro.59.050405.101230.

33. Clarke DJ. 2010. The Rcs phosphorelay: more than just a two-componentpathway. Future Microbiol 5:1173–1184. https://doi.org/10.2217/fmb.10.83.

34. Bode NJ, Debnath I, Kuan L, Schulfer A, Ty M, Pearson MM. 2015.Transcriptional analysis of the MrpJ network: modulation of diversevirulence-associated genes and direct regulation of mrp fimbrial and

flhDC flagellar operons in Proteus mirabilis. Infect Immun 83:2542–2556.https://doi.org/10.1128/IAI.02978-14.

35. Meier-Dieter U, Starman R, Barr K, Mayer H, Rick PD. 1990. Biosynthesisof enterobacterial common antigen in Escherichia coli. Biochemical char-acterization of Tn10 insertion mutants defective in enterobacterial com-mon antigen synthesis. J Biol Chem 265:13490 –13497.

36. Claret L, Hughes C. 2000. Functions of the subunits in the FlhD2C2transcriptional master regulator of bacterial flagellum biogenesis andswarming. J Mol Biol 303:467– 478. https://doi.org/10.1006/jmbi.2000.4149.

37. Pearson MM, Yep A, Smith SN, Mobley HLT. 2011. Transcriptome ofProteus mirabilis in the murine urinary tract: virulence and nitrogenassimilation gene expression. Infect Immun 79:2619 –2631. https://doi.org/10.1128/IAI.05152-11.

38. Kuhn HM, Neter E, Mayer H. 1983. Modification of the lipid moiety of theenterobacterial common antigen by the “Pseudomonas factor.” InfectImmun 40:696 –700.

39. Kiss P, Rinno J, Schmidt G, Mayer H. 1978. Structural studies on the immu-nogenic form of the enterobacterial common antigen. Eur J Biochem88:211–218. https://doi.org/10.1111/j.1432-1033.1978.tb12440.x.

40. Dell A, Oates J, Lugowski C, Romanowska E, Kenne L, Lindberg B. 1984.The enterobacterial common-antigen, a cyclic polysaccharide. Carbo-hydr Res 133:95–104. https://doi.org/10.1016/0008-6215(84)85186-1.

41. Rick PD, Mayer H, Neumeyer BA, Wolski S, Bitter-Suermann D. 1985.Biosynthesis of enterobacterial common antigen. J Bacteriol 162:494 –503.

42. Duda KA, Duda KT, Beczała A, Kasperkiewicz K, Radziejewska-Lebrecht J,Skurnik M. 2009. ECA-immunogenicity of Proteus mirabilis strains. ArchImmunol Ther Exp (Warsz) 57:147–151. https://doi.org/10.1007/s00005-009-0018-9.

43. Kunin CM. 1963. Separation, characterization, and biological significanceof a common antigen in Enterobacteriaceae. J Exp Med 118:565–586.https://doi.org/10.1084/jem.118.4.565.

44. Whang HY, Neter E. 1962. Immunological studies of a heterogeneticenterobacterial antigen (Kunin). J Bacteriol 84:1245–1250.

45. Fomsgaard A, Freudenberg MA, Galanos C. 1990. Modification of thesilver staining technique to detect lipopolysaccharide in polyacrylamidegels. J Clin Microbiol 28:2627–2631.

46. McCoy AJ, Liu H, Falla TJ, Gunn JS. 2001. Identification of Proteusmirabilis mutants with increased sensitivity to antimicrobial peptides.Antimicrob Agents Chemother 45:2030 –2037. https://doi.org/10.1128/AAC.45.7.2030-2037.2001.

47. Merritt ME, Donaldson JR. 2009. Effect of bile salts on the DNA andmembrane integrity of enteric bacteria. J Med Microbiol 58:1533–1541.https://doi.org/10.1099/jmm.0.014092-0.

48. Ramos-Morales F, Prieto AI, Beuzon CR, Holden DW, Casadesus J. 2003.Role for Salmonella enterica enterobacterial common antigen in bileresistance and virulence. J Bacteriol 185:5328 –5332. https://doi.org/10.1128/JB.185.17.5328-5332.2003.

49. Elßner T, Preußer A, Wagner U, Kleber HP. 1999. Metabolism of L(�)-carnitine by Enterobacteriaceae under aerobic conditions. FEMS MicrobiolLett 174:295–301. https://doi.org/10.1111/j.1574-6968.1999.tb13582.x.

50. Eichler K, Buchet A, Lemke R, Kleber HP, Mandrand-Berthelot MA. 1996.Identification and characterization of the caiF gene encoding a potentialtranscriptional activator of carnitine metabolism in Escherichia coli. J Bacte-riol 178:1248–1257. https://doi.org/10.1128/jb.178.5.1248-1257.1996.

51. Engemann C, Kleber HP. 2001. Epigenetic regulation of carnitine metabolis-ing enzymes in Proteus sp. under aerobic conditions. FEMS Microbiol Lett196:1–6. https://doi.org/10.1111/j.1574-6968.2001.tb10531.x.

52. Landfald B, Strøm AR. 1986. Choline-glycine betaine pathway confers ahigh level of osmotic tolerance in Escherichia coli. J Bacteriol 165:849 – 855. https://doi.org/10.1128/jb.165.3.849-855.1986.

53. Beumer RR, Giffel Te MC, Cox LJ, Rombouts FM, Abee T. 1994. Effect ofexogenous proline, betaine, and carnitine on growth of Listeria mono-cytogenes in a minimal medium. Appl Environ Microbiol 60:1359 –1363.

54. Meadows JA, Wargo MJ. 2015. Carnitine in bacterial physiology andmetabolism. Microbiology 161:1161–1174. https://doi.org/10.1099/mic.0.000080.

55. Xiao M, Lai Y, Sun J, Chen G, Yan A. 2016. Transcriptional regulation ofthe outer membrane porin gene ompW reveals its physiological roleduring the transition from the aerobic to the anaerobic lifestyle ofEscherichia coli. Front Microbiol 7:799. https://doi.org/10.3389/fmicb.2016.00799.

56. Castaño-Cerezo S, Bernal V, Post H, Fuhrer T, Cappadona S, Sánchez-Díaz

Little et al. Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 16

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

NC, Sauer U, Heck AJR, Altelaar AFM, Cánovas M. 2014. Protein acetyla-tion affects acetate metabolism, motility and acid stress response inEscherichia coli. Mol Syst Biol 10:762. https://doi.org/10.15252/msb.20145227.

57. Lane MC, Li X, Pearson MM, Simms AN, Mobley HLT. 2009. Oxygen-limiting conditions enrich for fimbriate cells of uropathogenic Proteusmirabilis and Escherichia coli. J Bacteriol 191:1382–1392. https://doi.org/10.1128/JB.01550-08.

58. Yao Z, Kahne D, Kishony R. 2012. Distinct single-cell morphologicaldynamics under beta-lactam antibiotics. Molecular Cell 48:705–712.https://doi.org/10.1016/j.molcel.2012.09.016.

59. Ranjit DK, Young KD. 2013. The Rcs stress response and accessoryenvelope proteins are required for de novo generation of cell shape inEscherichia coli. J Bacteriol 195:2452–2462. https://doi.org/10.1128/JB.00160-13.

60. Anderson-Furgeson JC, Zupan JR, Grangeon R, Zambryski PC. 2016. Lossof PodJ in Agrobacterium tumefaciens leads to ectopic polar growth,branching, and reduced cell division. J Bacteriol 198:1883–1891. https://doi.org/10.1128/JB.00198-16.

61. de Pedro MA, Young KD, Höltje J-V, Schwarz H. 2003. Branching of Esche-richia coli cells arises from multiple sites of inert peptidoglycan. J Bacteriol185:1147–1152. https://doi.org/10.1128/JB.185.4.1147-1152.2003.

62. Nilsen T, Ghosh AS, Goldberg MB, Young KD. 2004. Branching sites andmorphological abnormalities behave as ectopic poles in shape-defectiveEscherichia coli. Mol Microbiol 52:1045–1054. https://doi.org/10.1111/j.1365-2958.2004.04050.x.

63. Potluri L-P, de Pedro MA, Young KD. 2012. Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and theirabsence leads to asymmetric cell division and branching. Mol Microbiol84:203–224. https://doi.org/10.1111/j.1365-2958.2012.08023.x.

64. Zupan JR, Cameron TA, Anderson-Furgeson J, Zambryski PC. 2013.Dynamic FtsA and FtsZ localization and outer membrane alterationsduring polar growth and cell division in Agrobacterium tumefaciens. ProcNatl Acad Sci U S A 110:9060 –9065. https://doi.org/10.1073/pnas.1307241110.

65. Pannen D, Fabisch M, Gausling L, Schnetz K. 2016. Interaction of the RcsBresponse regulator with auxiliary transcription regulators in Escherichiacoli. J Biol Chem 291:2357–2370. https://doi.org/10.1074/jbc.M115.696815.

66. Belas R, Erskine D, Flaherty D. 1991. Transposon mutagenesis in Proteus

mirabilis. J Bacteriol 173:6289 – 6293. https://doi.org/10.1128/jb.173.19.6289-6293.1991.

67. Gibbs KA, Urbanowski ML, Greenberg EP. 2008. Genetic determinants ofself identity and social recognition in bacteria. Science 321:256 –259.https://doi.org/10.1126/science.1160033.

68. Kaniga K, Delor I, Cornelis GR. 1991. A wide-host-range suicide vector forimproving reverse genetics in Gram-negative bacteria: inactivation ofthe blaA gene of Yersinia enterocolitica. Gene 109:137–141. https://doi.org/10.1016/0378-1119(91)90599-7.

69. Saak CC, Gibbs KA. 2016. The self-identity protein IdsD is communicatedbetween cells in swarming Proteus mirabilis colonies. J Bacteriol 198:3278 –3286. https://doi.org/10.1128/JB.00402-16.

70. Zhang Y, Werling U, Edelmann W. 2012. SLiCE: a novel bacterial cellextract-based DNA cloning method. Nucleic Acids Res 40:e55. https://doi.org/10.1093/nar/gkr1288.

71. Guzman LM, Belin D, Carson MJ, Beckwith J. 1995. Tight regulation,modulation, and high-level expression by vectors containing the arabi-nose PBAD promoter. J Bacteriol 177:4121– 4130. https://doi.org/10.1128/jb.177.14.4121-4130.1995.

72. Balleza E, Kim JM, Cluzel P. 2018. Systematic characterization of matu-ration time of fluorescent proteins in living cells. Nat Methods 15:47–51.https://doi.org/10.1038/nmeth.4509.

73. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T,Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ,Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676 – 682.https://doi.org/10.1038/nmeth.2019.

74. Sambrook J, Russell DW. 2006. Purification of nucleic acids by extractionwith phenol:chloroform. CSH Protoc 2006:pdb.prot4455. https://doi.org/10.1101/pdb.prot4455.

75. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. 2013.TopHat2: accurate alignment of transcriptomes in the presence of in-sertions, deletions and gene fusions. Genome Biol 14:R36. https://doi.org/10.1186/gb-2013-14-4-r36.

76. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L.2013. Differential analysis of gene regulation at transcript resolutionwith RNA-seq. Nat Biotechnol 31:46 –53. https://doi.org/10.1038/nbt.2450.

77. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. 2016. KEGGas a reference resource for gene and protein annotation. Nucleic AcidsRes 44:D457–D462. https://doi.org/10.1093/nar/gkv1070.

Swarmer Cell Development Requires the rffG Gene Journal of Bacteriology

September 2018 Volume 200 Issue 18 e00230-18 jb.asm.org 17

on August 1, 2020 by guest

http://jb.asm.org/

Dow

nloaded from