1.                  Introduction

Bacterial growth and morphology are regulated by peptide glycan synthesis complexes and associated membrane proteins [1]. The chromosomal region at 14 min on the Escherichia colichromosome map (the mrd region) contains several genes involved in cell duplication and morphogenesis [2]. These include mrdA[3] (also called pbpA[4]), which codes for the penicillin-binding protein PBP-2;mrdB[3] (also calledrodA[5]), which codes for the RodA protein; anddacA[6], which codes for PBP-5.PBP-2 and RodA are involved in determination of the rod shape of the E. colicell, functioning in the biosynthesis of peptidoglycan during cell growth [7]. PBP-5 is a D-alanine carboxypeptidase involved in the maturation of peptidoglycan [6].

Previously, we identified two rare lipoprotein genes, rlpA and rlpB (also called lptE), in the mrd region [8]. RlpA is located between mrdB and dacA and encodes a 36-kDa lipoprotein, while rlpB/lptE is located several kilobases upstream from mrdA/pbp2 and encodes a 19-kDa lipoprotein. These lipoproteins localize to the OuterMembrane (OM), and their maturation is inhibited by globomycin, an inhibitor of signal peptidase for the major E. coliLipoprotein (Lpp) and other lipoproteins [9,10]. Though rlpAandrlpB/lptEappear to be expressed at low levels, their product lipoproteins maystill be functionally important for the cell.

2.                  Materials and Methods

Recent studies have revealed that the RlpB/LptE lipoprotein forms the LptD-LptE complex, which is responsible for inserting lipopolysaccharide into the outer leaflet of the OM [11-15]. The RlpA lipoprotein localizes to the ring-shaped apparatus called the septal ring, which mediates bacterial cytokinesis by binding to septal peptide glycan through the C-terminal SPOR domain [16-18]. The E. colichromosome encodes four SPOR domain proteins, namely FtsN, DamX, DedD, and RlpA, all of which localize to the septal ring. Mutation studies have revealed that FtsN, DamX, and DedD are cell division proteins and are involved in bacterial cytokinesis [16-19]. However, there is no evidence as yet that RlpA is involved in cell division and morphogenesis. Thus, the physiological role of RlpA remains unknown. To elucidate the physiological function of RlpA, we constructed an RlpA deletion mutant through homologous recombination. Double mutants lacking both the RlpA and Lpp lipoproteins were also constructed, and the effect of deleting rlpA was determined.

The E. colistrains used in this study are listed in Table 1. For the genetic experiments and the antibiotic sensitivity test, modified Lennox-broth, L' broth, were used, supplemented with 10 g/L polypeptone (DaigoEiyo Chemical Co., Osaka), 5 g/Lyeast extract, 5 g/L NaCl, 1 g/L glucose, 20 mg/L thymine, and 0.1 mg/L lipoic acid (L' agar). For the P1 phage transduction experiments, L'-agar plate containing 50 m g/ml kanamycin, and M9 agar plates supplemented with 50 mg/L appropriate amino acids, 1 mg/L thiamine and 0.1 mg/L lipoic acid were used. P1 phage transduction technique was performed as previously described [20].

To construct the rlpA deletion mutant (DrlpA), we adopted a homologous recombination method, using a temperature-sensitive plasmid, as reported by Matsuyama et al.[21]. A 15-kb fragment of the E. coli dacA11191[6] chromosome covering the region from leuS to dacA (mrdregion) was cloned into the temperature-sensitive plasmid pMAN031. The rlpAgene was then replaced with a kanamycin resistance (km^r) gene. The km^rgene was inserted in each direction, resulting in the two plasmids pHK002 and pHK003, each of which carried km^rin an opposite direction from the other (Figure 1(A)). E. coliJE1011 was transformed with these plasmids. Cells were then grown on L' agar plates containing 50 µg/mL kanamycin, and were incubated overnight at 42^°C. The colonies were replicated on an L' agar plate containing 50 µg/mL ampicillin and incubated overnight at 30^°C. Five out of 10³Km^rcolonies were also determined to be ampicillin sensitive (Amp^s). These Km^r Amp^scolonies are believed to be mutants lacking the rlpA gene from the chromosome.

Since the plasmids pHK002 and pHK003 contained the dacA11191mutant gene coding for a mutant PBP-5 with no penicillin-releasing activity [22]. We expected to obtain two types of mutants: DrlpAmutants with wild-type dacA and DrlpAdacA11191mutants. Previously, we found that theE. coli dacA11191 mutant was moresensitive to penicillins than the wild-type dacA strain [3]. To distinguish between wild-type dacA⁺ strains and dacA11191mutant strains, we measured the ampicillin sensitivities of the Km^r Amp^sstrains using a paper strip method. This allowed us to classify these strains as either ampicillin-sensitive or ampicillin-supersensitive. Two strains, E. coliEC2009 and EC3001, exhibited normal ampicillin sensitivity, whereas the other two strains, E. coliEC2001 and EC3002, exhibited ampicillin supersensitivity (Table 2). EC2001 and EC2009 were pHK002 transformants, and EC3001 and EC3002 were pHK003 transformants. We performed a penicillin-releasing assay [22] to confirm that the ampicillin-sensitive transformants expressed wild-type PBP-5, while the ampicillin-supersensitive transformants expressed the mutant PBP-5 (Figure 2)

The order of genes on the DrlpA mutant chromosome was determined bymapping of the km^rgene with P1 phage and by Southern blot hybridization analysis. P1 phage transduction technique was performed as previously described[20]. DrlpA mutation was transduced with the km^r gene as the marker using P1 phages grown in the DrlpAtransformants into the recipientE.coli lip^-AT1325. The co-transductionfrequencies of thekm^rgene and the lip gene were 79% in EC2009, 80% in EC3001, 78% in EC2001, and 74% in EC3002. These frequencies were similar to the co-transduction frequency (76%) of mrdB and lip in a previous study [3]. Deletion of the rlpA gene from the E. colichromosome was confirmed by Southern hybridization (Figure 1(B)).

Isolated DrlpA mutants grew normally in L' broth, exhibiting the normal rod shape. Omitting NaCl from the medium and growing cells at a higher temperature (42^°C) did not affect the growth or morphology of the DrlpAmutants. We detected no sign that the rlpAgene is indispensable for growth and shape-determination of the cell. Double mutant strains lacking both the RlpA and Lpp lipoproteins (BC2001 and BC3001) were isolated by transducing the lppmutant strain E. coliGRB19 with P1 phage grown in one of the E. coliDrlpA mutants (EC2009 or EC3002) using the km^rgene as a transduction marker. Triple mutants BCA2001 and BCA3001, carrying DrlpAlpp dacA11191, were isolated by transduction ofE. coli GRB19 with P1 phage grown inE. coli EC2001 or EC3002. All mutants with both lipoprotein deletions exhibited thenormal rod shape.

Despite exhibiting normal morphologies, the DrlpA mutants showed increased sensitivity to several antibiotics, as measured by paper strip and step dilution methods (Table 2). Vancomycin and bacitracin were obtained commercially from Sigma (St. Louis, MO, USA); ampicillin, benzylpenicillin, and macarbomycin were obtained from Meiji-Seika Co. (Tokyo, Japan); moenomycin was obtained from Hoechst (Frankfurt, Germany); enramycin was obtained from Takeda Chemical Industries (Osaka, Japan); and globomycin was obtained from Sankyo Co. (Tokyo, Japan). Sensitivity to antibiotics such as moenomycin, macarbomycin, enramycin, vancomycin, and bacitracin increased appreciably when the DrlpA mutation was introduced into the JE1011 strain, and sensitivity to globomycin also increased slightly. In contrast, deletion of rlpA did not greatly affect sensitivity to penicillin, rifampicin, or novobiocin (data not shown). No increases in sensitivity to the above antibiotics have been observed due to introduction of the kanamycin resistance mutation alone (data notshown).

It is well known that SPOR-domain proteins are recruited to the septal ring, which mediates bacterial cytokinesis [1]. Among these proteins, FtsN is essential for cell division [19], and DamX and DedD are genuine division proteins that contribute significantly to the cell constriction process [16-18]. In contrast, the function of the SPOR-domain protein RlpA remains unclear. A recent study by Yahasiri et al. revealed that in Pseudomonas aeruginosa,RlpA is a lytic transglycosylase that contributes to rod shape and daughter cell separation [23]. Thus far, however, researchers have been unable to obtain any evidence indicating that the E. coliRlpA exhibits lytic transglycosylase activity [16]. We found that E. coli rlpA deletion mutants divided normally and did not show any significant morphological changes, consistent with previous findings [16-18].

3.                  Conclusion

Nevertheless, it is unlikely that E. coliRlpA is not involved in cell division or morphogenesis in some way. It has been reported that a truncated rlpA mutant is a multicopy suppressor of a mutation of the periplasmic protease gene prc, which is involved in cell division [24]. It is probable that double or multiple mutations in different lipoproteins are required to cause such phenotypic changes in the cell. Alternatively, RlpA may be involved in the membrane integrity of E. coli. RlpA localizes not only to the septal ring but also to foci at various sites along the cell cylinder, suggesting that RlpA plays distinct roles in cytokinesis and envelope maturation [17]. Sensitivity to globomycin increased slightly in the DrlpA mutants, while it has been shown to decrease in lppmutants [25]. As formation of the RlpA lipoprotein and its assembly at the membrane are inhibited by globomycin [8], the increased sensitivity of DrlpA mutants to globomycin may indicate that RlpA is involved in cell proliferation. In this model, RlpA would act as a minor component of membrane lipoproteins in the formation of the E. colicell envelope.

4.                  Acknowledgments

We regret the early, sudden death of our collaborator Dr. Ichiro Takase. This article is dedicated to his memory.

Figure 1:Isolation of DrlpA mutants by homologous recombination. (A) Homologous recombination of chromosome and plasmid led to deletion of therlpAgene and insertion of thekm^rgene into the chromosome. Arrows indicate thedirection of km^rgene transcription. Two types of rlpA deletion mutants were obtained from transformants according to the sites of recombination, one deriving fromrecombination between km^r and dacA and the other deriving from recombination between km^r and dacA11191. (B) Chromosomal DNAs were digested with BamHI (left) and BglII (right), and were subjected to southern blot hybridization with probe 1 (left) and probe 2 (right), respectively.

Figure 2: Penicillin-releasing assay of DrlpA mutants.Penicillin-binding proteins fromthe membrane fractions of E.coli strains were labeled with [¹⁴C]penicillin G, and the release of penicillin was analyzed by addition of unlabeled penicillin G. After various lengths of time, the reaction was stopped, and the samples were subjected to SDS-PAGE and gel fluorograms.

1.       Typas A, Banzhaf M, Gross CA, Vollmer W (2012) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol10:123-136.

2.       Berlyn MK (1998) Linkage map of Escherichia coli K-12, edition 10: the traditional map. MicrobiolMolBiol Rev62:814-984.

3.       Tamaki S, Matsuzawa H, Matsuhashi M (1980) Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli. J Bacteriol141:52-57.

4.       Spratt BG, Boyd A, Stoker N (1980) Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome. J Bacteriol143:569-581.

5.       Matsuzawa H, Hayakawa K, Sato T, Imahori K(1973) Characterization and genetic analysis of a mutant of Escherichia coli K-12 with rounded morphology. J Bacteriol115:436-442.

6.       Matsuhashi M, Maruyama IN, Takagaki Y, Tamaki S, Nishimura Y, et al. (1978) Isolation of a mutant of Escherichia coli lacking penicillin-sensitive D-alanine carboxypeptidase IA. Proc Natl Acad Sci USA75:2631-2635.

7.       Ishino F, Park W, Tomioka S, Tamaki S, Takase I, et al. (1986) Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein. J BiolChem261:7024-7031.

8.       Takase I, Ishino F, Wachi M, Kamata H, Doi M, et al. (1987) Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome. J Bacteriol 169:5692-5699.

9.       Braun V, Rehn K (1969) Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall. The specific effect of trypsin on the membrane structure. Eur J Biochem10:426-438.

10.    Ichihara S, Hussain M, Mizushima S (1981) Characterization of new membrane lipoproteins and their precursors of Escherichia coli. J BiolChem256:3125-3129.

11.    Dong H, Xiang Q, Gu Y, Wang Z, Paterson NG, et al. (2014) Structural basis for outer membrane lipopolysaccharide insertion. Nature511:52-56.

12.    Qiao S, Luo Q, Zhao Y, Zhang XC, Huang Y (2014) Structural basis for lipopolysaccharide insertion in the bacterial outer membrane. Nature511:108-111.

13.    Chng SS, Xue M, Garner RA, Kadokura H, Boyd D, et al. (2012) Disulfide rearrangement triggered by translocon assembly controls lipopolysaccharide export. Science337:1665-1668.

14.    Wu T, McCandlish AC, Gronenberg LS, Chng SS, Silhavy TJ, et al. (2006) Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli. Proc Natl Acad Sci U SA103:11754-11759.

15.    Malojcic G, Andres D, Grabowicz M, George AH, Ruiz N, et al. (2014)LptE binds to and alters the physical state of LPS to catalyze its assembly at the cell surface. Proc Natl Acad Sci USA111:9467-9472.

16.    Yahashiri A, Jorgenson MA, Weiss DS (2015) Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides. Proc Natl Acad Sci USA112:11347-11352.

17.    Gerding MA, Liu B, Bendezu FO, Hale CA, Bernhardt TG, et al. (2009) Self-enhanced accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR domain (DamX, DedD, and RlpA) in Escherichia coli cell constriction. J Bacteriol191:7383-7401.

18.    Arends SJ, Williams K, Scott RJ, Rolong S, Popham DL, et al. (2010) Discovery and characterization of three new Escherichia coli septal ring proteins that contain a SPOR domain: DamX, DedD, and RlpA. J Bacteriol192:242-255.

19.    Dai K, Xu Y, Lutkenhaus J (1993) Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts). J Bacteriol175:3790-3797.

20.    Tamaki S, NakagawaJ, Maruyama I, Matsuhashi M (1979) Supersensitivity to beta-lactam antibiotics in Escherichia coli caused by D-alanine carboxypeptidase IA mutation. Agric BiolChem42:2147-2150.

21.    Matsuyama S, Mizushima S (1985) Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli. J Bacteriol162:1196-1202.

22.    Matsuhashi M, Tamaki S, Curtis SJ, Strominger JL (1979) Mutational evidence for identity of penicillin-binding protein 5 in Escherichia coli with the major D-alanine carboxypeptidase IA activity. J Bacteriol137:644-647.

23.    Jorgenson MA, Chen Y, Yahashiri A, Popham DL, Weiss DS (2014) The bacterial septal ring protein RlpA is a lytic transglycosylase that contributes to rod shape and daughter cell separation in Pseudomonasaeruginosa. MolMicrobiol93:113-128.

24.    Bass S, Gu Q, Christen A (1996) Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA. J Bacteriol178:1154-1161.

25.    Inukai M, Takeuchi M, Shimizu K, Arai M (1978) Mechanism of action of globomycin. J Antibiot (Tokyo)31:1203-1205.

26.    Taylor AL, Thoman MS (1964) The Genetic Map of Escherichia coliK-12. Genetics 50:659-677.