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Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB, yojN or rcsC strains of Salmonella enterica serovar Typhimurium

Cristina S. Costa, M. Julia Pettinari, Beatriz S. Méndez, Dora N. Antón
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00221-0 25-32 First published online: 1 May 2003

Abstract

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.

Keywords
  • yrfF
  • mucM
  • igaA
  • Essential gene
  • Mucoid mutant
  • rcsC-yojN-rcsB phosphorelay

1 Introduction

It has recently been reported that the most frequent type of mucoid mutant isolated by resistance to mecillinam in Salmonella enterica serovar Typhimurium is affected in a new gene, tentatively called mucM, located at about centisome (Cs) 76, between markers aroB and envZ [1]. Mucoidy and resistance of mucM mutants to mecillinam have been assigned to increased activity of gene rcsB. rcsB product is the positive effector of a complex phosphorelay system formed by genes rcsC, yojN, and rcsB that regulates the transcription of a cluster of 19 genes (cps genes) involved in the synthesis of capsule exopolysaccharide colanic acid [2,3]. Moreover, RcsB activates transcription of the cell division genes ftsZ and ftsA by acting specifically on one of the promoters (ftsA1p) governing expression of the ftsQAZ cluster [4]. Increased transcription of cps genes in mucM mutants would lead to mucoidy while overexpression of FtsZ and FtsA proteins would result in resistance to mecillinam [1]. In this paper, mucM was found to be the gene designated by McClelland et al. [5] as yrfF, and results demonstrating that the yrfF gene product is essential to wild-type S. typhimurium but is not required for survival of rcsB, yojN, or rcsC mutants are presented.

2 Materials and methods

2.1 Bacterial strains, phage and media

All the strains used were derivatives of S. typhimurium LT2 unless otherwise specified; their origins and relevant genetic properties appear in Table 1. All the strains carrying plasmids were grown in LB broth containing the appropriate antibiotic to keep the plasmid; strains carrying plasmid pCC74 were grown at 25°C for better maintenance of the plasmid. Transductions were mediated by phage P22 HT105/1 int-201 as previously described [1]. Complete medium was LB broth and LB agar, and E medium was used as minimal medium [6]. MacConkey agar was used to test fermentation of lactose. Antibiotics were used at the following concentrations: mecillinam, 1 µg ml−1; kanamycin, 20 µg ml−1; tetracycline, 20 µg ml−1; ampicillin, 100 µg ml−1. Mecillinam was a kind gift of Leo Pharmaceutical Products (Denmark).

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Table 1

Bacterial strains and plasmids used in this work

Strain or plasmidRelevant characteristicsOrigin
Strain
DA1468argC95[1]
DA1736cysB484Laboratory collection
DA1952zhg-7131::Tn10dTet[1]
DA2023recA1 zfj-623::Tn10dCamLaboratory collection
DA2025yrfF1[1]
DA2026yrfF1 wcaJ1::MudJ[1]
DA2032yrfF9::MudJ wcaJ1::MudJThis work
DA2044wcaJ1::MudJ[1]
DA2069yojN1::Tn10dTetThis work
DA2070rcsC37::Tn10dTetThis work
DA2092aroB25Laboratory collection
DA2095yrfF9::MudJ rcsC29This work
DA2103yrfF28::Kan rcsC37::Tn10dTetThis work
DA2108rcsB15::Tn10dTetThis work
DA2109yrfF1 rcsB15::Tn10dTetThis work
DA2112yrfF28::Kan rcsB30This work
DA2118yrfF1 yojN1::Tn10dTetThis work
DA2119yrfF1 rcsC37::Tn10dTetThis work
DA2120cps-4::Tn10dTetThis work
SH7241ompC396::Tn10[16]
TT5371zef-754::Tn10[16]
TT10289hisD9953::MudJ his-9949::MudI[13]
TT15258cysA1586::MudQ[9]
TT15265envZ1005::MudP[9]
Plasmid
pGEM-T EasyCloning vector, Amp-RPromega
pWSK29Cloning vector, Amp-R[10]
pUC4KSource of Kan-R cassettePharmacia
pCC14pGEM-T Easy+yrfE+This work
pCC23pGEM-T Easy+yrfF+This work
pCC24Derived from pCC23, yrfF28::KanThis work
pCC25pGEM-T Easy+rcsB+This work
pCC26pWSK29+rcsB+This work
pCC74pGEM-T Easy+yojN+This work
pFAB4Transcriptional fusion ftsA1p-lacZ[4]
  • All the DA strains are derivatives of DA1468 and contain the argC95 mutation.

2.2 Isolation and characterization of rcsB, yojN and rcsC insertion mutants

Insertional mutations in genes rcsB, yojN and rcsC were obtained by transducing strain DA2026 (yrfF1 wcaJ1::MudJ) with phage grown on a pool of random Tn10dTet insertions. Tetracycline-resistant (Tetr) transductants that produced white colonies on MacConkey plus tetracycline agar were characterized by transduction and polymerase chain reaction (PCR). Insertions that showed cotransduction with marker gyrA were identified as affecting the yojN-rcsB-rcsC group. DNA of those mutants was used for amplification with the following primers: UrcsB (5′-GAA GAG ATT CCC GCC TCC C-3′) and LrcsB (5′-GTG TAT GCC GAG CGG GTA CG-3′) for rcsB, UyojN (5′-CGG CTG ATT TAT GCT ACC TG-3′) and LyojN (5′-GTA CAA TCG GGT GGT CAT CG-3′) for yojN, and UrcsC (5′-CGC TTT TAT GTT ACC CAG CC-3′) and LrcsC (5′-GTT ATT GCT GTG CGA GGG-3′) for rcsC. Size modification and change in the restriction pattern of the amplified fragments due to insertion of the 2910-bp transposon allowed identification of the gene affected by Tn10dTet insertion. Among five strains analyzed, one rcsB, one rcsC and three yojN mutants were identified.

2.3 Recombinant DNA techniques

Plasmids used in this work are listed in Table 1. Conventional recombinant DNA techniques were used for manipulation of DNA, amplification by PCR and plasmid construction [7]. Primers used for PCR were designed on the basis of the genome sequence of S. typhimurium LT2 [5]. Escherichia coli DH5α (Bethesda Research Laboratories) was used as recipient strain for plasmid constructions that were, subsequently, transformed into S. typhimurium DA2023 (recA1), and then transferred by transduction to other S. typhimurium strains.

2.4 Inverse PCR

DNA flanking the left end of yrfF9::MudJ was obtained as described by Ahmer et al. [8]. Genomic DNA of strain DA2095 was isolated and digested with enzymes TaqI or AluI. Different aliquots of the restricted DNA were treated with T4 DNA ligase and inverse PCR was performed using these ligations as templates. DNA digested with TaqI was amplified with primers MudTaq and MudOut [8]; DNA digested with AluI was amplified with primers MudAlu and MudOut [8]. An amplification product of 386 bp, obtained with TaqI-digested DNA, was cloned into plasmid pGEM-T Easy (Promega) and sequenced. The same procedure was used with strain DA2044 (wcaJ1::MudJ) to identify the cps gene affected in the fusion formerly called cps-1::MudJ [1]. In this case, sequence analysis of an amplification product of 650 bp obtained with AluI-digested DNA showed MudJ to be inserted in gene wcaJ.

2.5 Construction of plasmids

Genes yrfF and yrfE were obtained by PCR amplification of DNA from strain TT15265 enriched in the Cs 76 chromosomal region of S. typhimurium [9]. The primers used were: UyrfF (5′-CGA CGG TTT CCA CTT TCA GA-3′) and LyrfF (5′-TCC AGC AAA ACG GTA TCC AC-3′) for amplification of yrfF, and UyrfE (5′-ATT GTG TTT AGC GGA TGA CG-3′) and LyrfE (5′-CGG GTG ATG GAG TGG GTT AG-3′) for amplification of yrfE. The 2615-bp (yrfF) and 1243-bp (yrfE) amplification fragments were cloned into pGEM-T Easy, to obtain plasmids pCC23 and pCC14, respectively. The intergenic region between yrfF and yrfE was amplified from genomic DNA of the yrfF1 strain DA2025 using primers UyrfF and LyrfE and was cloned into pGEM-T Easy. For the construction of the yrfF28::Kan mutant, plasmid pUC4K (Pharmacia) was used as the source of a kanamycin resistance cassette (Kan) by digestion with BamHI. Introduction of the Kan cassette into the unique BamHI site of the yrfF gene in pCC23 produced plasmid pCC24 (Fig. 1).

Figure 1

Schematic representation of the S. typhimurium yrfE-yrfF region. Arrows represent the different ORFs and the orientation of transcription. Plasmids constructed for this study are represented as black lines indicating the DNA region carried by each plasmid. The location of the MudJ factor in the yrfF9 mutant and the restriction site used for insertion of the Kan cassette in the yrfF gene (yrfF28::Kan) are indicated by inverted triangles.

Primers UrcsB and LrcsB (see above) were used for amplification of the rcsB gene from genomic DNA of strain DA1468, the standard wild-type strain used in this laboratory [1]. The 1100-bp amplification product was cloned into multicopy vector pGEM-T Easy to obtain plasmid pCC25. Plasmid pCC25 produced mucoidy in all the strains tested, including wild-type DA1468. Therefore, to have gene rcsB in a low copy number plasmid, pCC25 was digested with EcoRI and the fragment containing rcsB was cloned into the EcoRI site of plasmid pWSK29 [10] to obtain plasmid pCC26. Primers UyojN and LyojN (see above) were used for amplification of gene yojN from DNA of strain TT15258 enriched in the Cs 50 chromosomal region of S. typhimurium [9]. The amplification fragment of 3015 bp obtained was cloned into vector pGEM-T Easy to produce plasmid pCC74.

2.6 Other methods

β-Galactosidase activity of wcaJ-1::MudJ and pFAB4 strains was assayed in stationary cultures grown in LB broth as described by Miller [11] on cells treated with sodium dodecyl sulfate and chloroform. Specific activities are expressed in Miller units referred to OD650.

The minimum inhibitory concentration (MIC) of mecillinam was determined by plating cells from stationary cultures on LB plates containing twofold serial dilutions of the antibiotic [12].

3 Results

3.1 Isolation of mutant mucM9::MudJ

About 70% of the mucoid mutants isolated by resistance to mecillinam belong to the new class mucM [1]. Many attempts to isolate mucM mutants by insertion of the Tn10dTet transposon into the mucM gene were carried out but all of them failed. Attempts to isolate mucM mutants by insertion of the MudJ factor [13] also failed until a mucoid mutant was finally obtained when strain DA1736 treated with a lysate grown on strain TT10289 [13] was plated on kanamycin plates and filter paper disks containing 1 µg of mecillinam were placed on the surface. There was no growth on a zone of about 3 cm diameter around the disk while on the rest of the plate, where only kanamycin was present, many non-mucoid MudJ transductants appeared. In just one case, a mucoid colony was observed in the inhibition zone near the mecillinam disk. This isolate displayed resistance to 80 µg ml−1 of mecillinam; its kanamycin resistance (Kanr) marker was 100% linked to the mucoid phenotype and was cotransduced with markers aroB (69%), cysG (33%), and zhg-7131::Tn10dTet (91%). It was concluded that the mutation was caused by insertion of MudJ into the mucM gene, and it was named mucM9::MudJ. No increase in β-galactosidase-specific activity due to the MudJ factor was observed in mucM9::MudJ strains; so, either the insertion was not in frame or the gene was not expressed.

3.2 Identification of gene mucM

In order to identify gene mucM, inverse PCR was performed using genomic DNA of DA2095, a mucM9::MudJ strain (Section 2). The MudJ factor was found to be inserted in the intergenic region between genes yrfE and yrfF, 166 bp from the yrfE origin and 123 bp from the yrfF origin (Fig. 1). As yrfE and yrfF are divergent genes whose transcription starts from the same intergenic region [5], mucM9::MudJ insertion could affect one or both of the two genes. To discern which of these genes was responsible for the mucM phenotype, yrfE and yrfF sequences, both of them including the intergenic region, were amplified by PCR and independently cloned in multicopy plasmid pGEM-T Easy (Fig. 1).

The plasmid carrying the wild-type yrfF gene (pCC23) was found to complement the mucoid phenotype of several mucM mutants of S. typhimurium (mucM1, mucM5, mucM8, mucM9::MudJ) and also of a mucM mutant of E. coli K-12. Transcription of the cps cluster in a mucM1 mutant carrying fusion wcaJ1::MudJ (DA2026) was also normalized by pCC23 (Table 2). A similar result was observed when the mucM9::MudJ strain (DA2032) was tested, although in this case the level of β-galactosidase activity remained somewhat higher than the wild-type level. On the other hand, the plasmid carrying the yrfE+ gene (pCC14) not only did not modify the mucoidy of mucM mutants, but promoted a significant increase in the β-galactosidase activity of wcaJ1::MudJ derivatives of mucM1 and mucM9::MudJ strains. This effect was due to plasmid pGEM-T Easy, because the plasmid with no insert also increased the activity of the enzyme but only in strains whose β-galactosidase activity was already increased (Table 2). The reason for this behavior is not known.

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Table 2

Effect of plasmids pCC23 (yrfF+) and pCC14 (yrfE+) on the expression of fusion wcaJ1::MudJ in mucM strains

StrainPlasmidInsert in plasmidβ-Galactosidase activity
yrfF1384±34
yrfF1pGEM-T Easy825±13
yrfF1pGEM-T EasyyrfF+1.6±0.2
yrfF1pGEM-T EasyyrfE+803±69
yrfF9::MudJ302±43
yrfF9::MudJpGEM-T Easy482±52
yrfF9::MudJpGEM-T EasyyrfF+21±2.4
yrfF9::MudJpGEM-T EasyyrfE+458±25
yrfF+2.1±0.1
yrfF+pGEM-T Easy3.2±1.5
yrfF+pGEM-T EasyyrfF+2.3±0.2
yrfF+pGEM-T EasyyrfE+2.6±0.8
  • The strains used were: DA2026 (yrfF1), DA2032 (yrfF9::MudJ) and DA2044 (yrfF+); all the strains carried fusion wcaJ1::MudJ (formerly called cps-1::MudJ [1]). β-Galactosidase activity is expressed in Miller units referred to OD650±S.D. [11]. All the assays were performed in duplicate and the results are averages of at least three independent experiments.

On the basis of the complementation observed with the plasmid carrying the yrfF+ insert, the name of mucM mutants was changed to yrfF mutants.

To find out whether other yrfF mutants also carried alterations in the yrfE-yrfF intergenic region, the intergenic region of the yrfF1 mutant was amplified by PCR and sequenced. No change was observed with respect to the wild-type sequence. It seems, then, that yrfF1 does not affect the yrfF regulatory region but rather the yrfF open reading frame (ORF) since the pCC23 plasmid also complemented it (Table 2).

3.3 Effects of a null yrfF mutation

In order to investigate the role of the yrfF gene, a null yrfF mutation was constructed by inserting the Kan cassette of plasmid pUC4K in the unique BamHI site of the yrfF ORF carried by plasmid pCC23 (Fig. 1). Transfer of the new plasmid, pCC24, to several yrfF mutants (yrfF1, yrfF5, yrfF8, yrfF9::MudJ) demonstrated that the modified yrfF gene (yrfF28::Kan) had lost the capacity to complement the mucoid phenotype of those mutants. To achieve integration of yrfF28::Kan into the chromosome, phage grown on a strain carrying the pCC24 plasmid was used to select for aroB+ Kanr transductants with appropriate aroB25 recipients, taking advantage of the close linkage between aroB and yrfF. It was observed that when the aroB25 recipient (DA2092) carried the wild-type rcsBC alleles, all of the aroB+ Kanr transductants were also resistant to ampicillin due to the presence of plasmid pCC24. However, when the aroB25 recipient also carried an rcsB or rcsC mutation, aroB+ Kanr transductants displaying sensitivity to ampicillin and containing no plasmid were easily obtained. Thus, in the latter case allelic exchange occurred and the yrfF28::Kan marker was integrated in the chromosome. All those yrfF28::Kan transductants grew quite normally and were non-mucoid. Phage grown on a yrfF28::Kan rcsB30 strain (DA2112) was used to transduce the yrfF28::Kan mutation to different recipient strains by selection for Kanr transductants. It was found that transduction of the yrfF28::Kan mutation to wild-type recipients produced a few normal-sized non-mucoid colonies and a large number of very tiny, almost transparent, colonies that failed to grow when restreaked on the same medium (Fig. 2a). Only large normal colonies were observed when the recipient of the yrfF28::Kan marker was an rcsC strain (Fig. 2b). Since both rcsB and rcsC are components of the phosphorelay system controlling transcription of the cps cluster [2], the capacity of rcsB and rcsC mutations to suppress the lethality of yrfF28::Kan could be due to their blocking effect on colanic acid synthesis. To test this possibility, strain DA2120 carrying insertion cps-4::Tn10dTet, which prevents colanic acid synthesis, was used as recipient for the yrfF28::Kan mutation. The cps-4::Tn10dTet mutation not only did not suppress yrfF28::Kan lethality but decreased even more the size of the abortive colonies (Fig. 2c). Similar results were observed when synthesis of colanic acid was stopped by a galE mutation (galE1922), and also when the recipient strain carried an rcsA mutation (rcsA16::Tn10dTet) that reduced expression of the cps cluster [14] (not shown). In all these cases, the simultaneous presence of an rcsC mutation in the recipient normalized the number and size of yrfF28::Kan transductants demonstrating that colanic acid synthesis per se was not responsible for lethality.

Figure 2

Colony and cell morphology of yrfF28::Kan derivatives. Insertion yrfF28::Kan was transduced to different recipients strains and the colonies that appeared on the transduction plates were photographed (a–c). Cells from those colonies were suspended in saline solution and phase contrast photomicrographed (d–f). Wild-type recipient (DA1468): a and d; rcsC37::Tn10dTet recipient (DA2070): b and e; cps-4::Tn10dTet recipient (DA2120): c and f. Bar represents 10 µm (d–f).

Abortive yrfF28::Kan colonies presented cellular heterogeneity: spherical cells, short filaments, as well as empty envelopes, and cellular debris. In many cases, dark round bodies could be seen at both ends of elongated and almost lysed cells (Fig. 2d). Cells from yrfF28::Kan cps-4::Tn10dTet colonies displayed severe anomalies too, and their general aspect was similar to that of yrfF28::Kan colonies, but there were many large ovoid cells and short lysed filaments containing regularly spaced dark bodies (Fig. 2f). Cells from yrfF28::Kan rcsC37::Tn10dTet colonies were normal bacilli (Fig. 2e).

yrfF is the first ORF in a presumptive operon formed also by three other genes: yrfG, yrfH (hslR), and yrfI (hslO), the latter two encoding heat shock proteins [5,15]. To verify that lethality was due to inactivation of yrfF rather than to a polar effect on transcription of downstream genes, wild-type recipients carrying plasmid pCC23 were transduced with yrfF28::Kan phage. It was found that the yrfF+ gene contained in plasmid pCC23 allowed those strains to produce normal colonies in contrast with the abortive colonies produced by the same strains lacking the plasmid. Thus, lethality was specifically due to loss of the yrfF function.

3.4 Effect of rcsB, rcsC, and yojN mutations on the phenotype of yrfF mutants

The effect was tested of mutations produced by insertion of the Tn10dTet transposon in genes rcsB (rcsB15::Tn10dTet), rcsC (rcsC37::Tn10dTet), and yojN (yojN1::Tn10dTet) on the phenotype of strains carrying either the yrfF28::Kan mutation or the viable yrfF1 allele. It was found that the three types of mutation allowed survival of yrfF28::Kan derivatives. In relation to yrfF1 strains, the three mutations behaved alike. They abolished mucoidy and reduced cps expression in the wcaJ1::MudJ yrfF1 strain from 384 units of β-galactosidase activity to 1.4 units in the corresponding rcsB, rcsC, or yojN derivatives. They also restored the sensitivity to mecillinam of the yrfF1 strain (MIC of mecillinam on the yrfF1 strain: 40 µg ml−1; MIC on any of the three derivatives: 0.08 µg ml−1) and normalized the expression of ftsZ and ftsA genes from the ftsA1p promoter (β-galactosidase activity of stationary cultures of strain yrfF1 carrying plasmid pFAB4 was 6900 units while the activity of derivatives of the same strain containing any one of the three suppressors was about 830 units). Plasmid pFAB4 carries a lacZ reporter gene fused to a fragment of the ftsQAZ promoter region comprising ftsA1p, the ftsZ promoter subjected to RcsB stimulation [4]. These results confirm that all those characteristics are dependent on every one of the three components of the rcsC-yojN-rcsB phosphorelay system.

3.5 Suppressors of yrfF28::Kan lethality

As described before, transduction of the yrfF28::Kan mutation to a wild-type recipient produced many abortive colonies and a few large ones. Further incubation of the transduction plates allowed spots of outgrowth to appear in many of the tiny colonies. Both large colonies and outgrowth spots produced normal non-mucoid colonies when restreaked on the same medium and appeared to be caused by secondary mutations which arose spontaneously and suppressed yrfF28::Kan lethality.

It was investigated whether survivors were double transductants which received both the yrfF28::Kan mutation and the permissive marker from the donor. To this purpose, wild-type strain DA1468 was transduced to Kanr with phage grown on a yrfF28::Kan rcsC37::Tn10dTet donor (DA2103) so that double transductants carrying the rcsC mutation of the donor would be marked by the resistance to tetracycline of the rcsC37::Tn10dTet insertion. Among 140 viable colonies that included large colonies appearing early and outgrowth spots appearing until 5 days after plating, only four (3%) carried the donor rcsC mutation and so were double transductants. Thus, the suppressor mutations carried by most of the survivors were not due to double transduction but to fresh mutations present in the recipient strain or produced on the selection plates.

Lethality suppressors from 43 yrfF28::Kan Tets transductants were subjected to genetic analysis to find out if they also affected rcsBC genes. To that purpose, the 43 strains were transduced to Tetr with phage grown on strains carrying either insertion ompC396::Tn10 (SH7241) or zef-754::Tn10 (TT5371), both of which are located at Cs 50 [16], very close to the yojN-rcsB-rcsC region. All 43 survivors produced Tetr transductants with zef-754::Tn10 phage but only 60% did the same with the ompC396::Tn10 phage, suggesting that in the latter case the wild-type allele of the lethality suppressor was so close to the selective marker that most of the ompC396::Tn10 transductants received it and died. Phage grown on Tetr derivatives of the 43 strains (either ompC396::Tn10 or zef-754::Tn10) was used to transduce the Tetr marker to the mucoid yrfF1 strain DA2025. In all cases, 50–90% of Tetr transductants became non-mucoid, demonstrating that the survivors carried mutations in the yojN-rcsB-rcsC region that suppressed expression of the cps cluster.

In order to identify the genes affected by the suppressor mutations, the complementing behavior of plasmids pCC26 (rcsB+, low copy) and pCC74 (yojN+, multicopy) was tested with yrfF+ and yrfF1 derivatives of rcsB15::Tn10dTet, yojN1::Tn10dTet and rcsC37::Tn10dTet mutants (Table 1). Plasmid pCC26 produced mucoidy in all the strains tested except in the wild-type strain DA1468 and in yrfF+ derivatives of rcsB mutants that remained non-mucoid. Plasmid pCC74 produced mucoidy in strain DA1468 as well as in yojN mutants, either yrfF+ or yrfF1, but yrfF+ and yrfF1 derivatives of rcsB and rcsC mutants remained non-mucoid. Thus, low-level RcsB turned cps expression on in yrfF+rcsC and yrfF+yojN mutants but not in yrfF+rcsB strains. On the other hand, high-level YojN protein led to mucoidy in yrfF+yojN strains but not in yrfF+rcsB or yrfF+rcsC mutants.

To obtain yrfF+ derivatives of the 43 suppressed strains, removal of the yrfF28::Kan mutation was effected by cotransducing the yrfF+ allele with close by insertion zhg-7131::Tn10dTet present in strain DA1952. Plasmids pCC26 (rcsB+) and pCC74 (yojN+) were then transduced to yrfF+ derivatives of the 43 strains. On the basis of the behavior of the two plasmids in well characterized rcsB, yojN and rcsC mutants and their behavior in each of the 43 strains, 14 (33%) of the suppressors were identified as yojN mutations, two (5%) were rcsB mutations, and 20 strains (46%) were probably rcsC mutants. The remaining seven suppressors (16%) produced atypical responses and appeared to be complex mutants carrying more than one mutation. Most of the strains producing few or no transductants with insertion ompC396::Tn10 carried mutations in gene yojN, the closest to that marker.

4 Discussion

The results presented in this paper identify gene yrfF as responsible for the mucM phenotype and demonstrate that it performs an essential function since its inactivation in wild-type strains leads to cell death. Insertion of MudJ in the intergenic region between the yrfF and yrfE genes produced mutation yrfF9::MudJ that displays the typical characteristics of mucM mutations even though only the level of transcription of the yrfF gene would be expected to be modified by the insertion. As full loss of the yrfF product is lethal for wild-type strains, it can be assumed that all the yrfF mutants isolated by resistance to mecillinam are partial mutants that maintain at least the minimal level of yrfF product or function compatible with survival. Although mucoidy makes yrfF mutants very conspicuous and facilitates isolation, the connection of rcsB, rcsC, and yojN genes with the essential role of YrfF appears to be unrelated to their known function as regulators of capsule synthesis. Since the lethality caused by yrfF loss was not relieved by mutations interfering with colanic acid synthesis nor by inactivation of RcsA, a short-lived protein required to optimize RcsB-positive regulation of cps genes [14], it can be concluded that cps genes do not participate in yrfF lethality. The same conclusion can be drawn from the absence of cps, galE, malA, and rcsA mutations among spontaneous suppressors of yrfF lethality. In fact, spontaneous suppressors included, almost exclusively, rcsC and yojN mutations. This leaves one to wonder if only selective advantage promoted the amazing abundance of these two types of mutants.

Evidence of the role of the rcsB-rcsC-yojN phosphorelay system in the regulation of other processes besides colanic acid synthesis is being gathered lately and has led to the proposal that it could act as a global regulator. Thus, its involvement in the synthesis of Vi antigen, flagellin, and invasion proteins in Salmonella typhi [17], in the control of swarming in E. coli and other bacteria [3], in the regulation of osmC gene expression [18], and in the recovery of cells treated with chlorpromazine [19] has been demonstrated. RcsB also works as a positive effector on ftsA1p, one of the promoters located upstream of the vital cell division gene ftsZ [4]. Results previously reported suggested that the resistance of yrfF mutants to mecillinam could be assigned to the overproduction of FtsZ and/or FtsA proteins promoted by increased RcsB activity. The abundance of small cells and short filaments observed in the abortive yrfF28::Kan colonies could perhaps reflect the increased level of FtsZ and FtsA cell division proteins caused by the enhanced activity of RcsB on ftsA1p promoter. It is well known that the FtsZ level is tightly regulated, and overproduction of this essential protein results in cell division anomalies [20]. A putative function as inner membrane protein has been assigned to the yrfF product on the basis of the identification of four hydrophobic transmembrane regions by sequence analysis [5]. Therefore, the observed severe structural damage and envelope fragility could also be assigned to the absence of YrfF from the inner cell membrane. However, as those anomalies did not appear in yrfF28::Kan strains lacking active RcsB even though their membranes were also deprived of YrfF, it is clear that the lethality of null yrfF mutations requires the action of the rcsB-rcsC-yojN group of genes to be manifested.

RcsC and YojN are inner membrane proteins [3,21] as YrfF is also supposed to be [5]. It has been proposed that the stimulus leading to activation of the rcsC-yojN-rcsB phosphorelay system is related to alterations of the cell envelope that are detected by RcsC [21], transmitted by YojN [3] and received by RcsB which, upon phosphorylation, becomes the positive effector of several regulatory processes. YrfF seems to act as some kind of negative regulator of RcsB activity, and the idea that RcsC (and/or YojN) could ‘feel’ environmental stimuli through interaction with YrfF, in such a way that mutations decreasing the YrfF amount or altering its properties would result in partial activation of RcsC whereas total loss of YrfF would cause full activation of the rcsC-yojN-rcsB system, is attractive. Lethality, in the latter case, would be evoked by RcsB action on some specific target. Only rcsC, yojN, and, at a much lower frequency, rcsB mutations were found among the spontaneous suppressors analyzed, and perhaps a more extensive screening might reveal infrequent types. Mutations rendering the target resistant to RcsB action could also be expected to act as suppressors of yrfF lethality, unless they themselves were lethal.

Similarity of YrfF to the UmoB protein of Proteus mirabilis has been reported (41% identity, 65% similarity) [22]. UmoB is a non-essential protein involved in the control of flagellar biogenesis, swarming and cell division [22]. Although no YrfF function is known yet, the fact that a yrfF mutant (named igaA) was obtained with a screen devised to isolate mutants of S. typhimurium able to proliferate in a fibroblast line [23] suggests a role connected with cell division. This role could be related to the increased activity of FtsZ in yrfF mutants [1].

Identification of yrfF as the gene responsible for the mucM phenotype correlates with observations reported in E. coli. Meberg et al. found that a deletion comprising mrcA, yrfE, and almost half of the yrfF gene caused mucoidy. That alteration was initially assigned to the deletion of gene mrcA that encodes penicillin binding protein 1a, but it was recently found that a deletion affecting only mrcA did not display those characteristics [24]. The traits reported clearly denote yrfF damage; yet, it is surprising that such an extended deletion caused mucoidy but was not lethal. This leaves one to wonder if loss of the other two genes affected by the deletion (mrcA and/or yrfE) suppressed yrfF lethality or yrfF is not essential in E. coli K-12.

Acknowledgments

The technical assistance of Ana Marı́a Flores, Susana Boretto and Matilde Flores is gratefully acknowledged. This work was supported in part by a grant of Fundación Balseiro, Argentina.

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