(1)  Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, USA

Received: 15 March 2001 / Accepted: 6 July 2001 / Published online: 22 August 2001

ABSTRACT

The bipA gene encodes a ribosome-associated GTPase postulated to be involved in regulatory functions in enteropathogenic Escherichia coli. Previous studies demonstrated that BipA is tyrosine phosphorylated in EPEC strains, but not in E. coli strain K12. Results presented here indicate that BipA function is required at low temperatures in E. coli K12, suggesting a regulatory role independent of phosphorylation and of pathogenicity.

Keywords. bipA - yihK - Cold sensitivity - Escherichia coli K12

INTRODUCTION

While examining our laboratory derivatives of Escherichia coli strain K12 (hereafter referred to as "strains", although all are mutant derivatives of E. coli strain K12), we discovered that our stock of strain D10 (Hfr21 relA1 spoT1 metB1 rna-10; Gesteland 1966) was cold sensitive. The bacteria grew apparently normally on LB agar plates incubated at 37°C, but failed to form single colonies when streaked on LB plates and incubated at 20°C (Fig. 1A). Comparison of growth kinetics in liquid cultures demonstrated that D10 exhibited a decreased growth rate at 20°C relative to other strains such as MG1655 (Fig. 1B). Cold sensitivity has not been reported previously for D10, therefore we sought to identify the genetic basis for this phenotype.

Fig. 1A-C. Strains D10 and AF600 (MG1655 bipA::kan) are cold sensitive. A The indicated strains were streaked on LB agar plates and incubated at 20°C for 4 days. B Strains were grown overnight at 37°C in LB broth, then diluted to a starting A₆₀₀ of approximately 0.02 (again in LB broth). Growth was allowed to proceed at 20°C in a BioScreen C Microbiology Reader from Labsystems as described previously (Flower 2001). Measurements were taken every 30 min, but for clarity only readings for each 5-h increment are shown. C Bacteria were grown overnight at 37°C, then subcultured to a starting A₆₀₀ equal to approximately 0.02 in LB broth. Cultures were incubated in the BioScreen at 37°C and readings were taken every 30 min. Symbols: closed squares, MG1655; open squares, AF600; closed triangles, D10 (E. coli Genetic Stock Center); open triangles, D10 (Flower laboratory stock)

RESULTS AND DISCUSSION

Isolation of multicopy suppressors of the cold sensitivity of strain D10

To ascertain the cause of the observed cold sensitivity of strain D10, we isolated multicopy suppressors from a genomic library. DNA was prepared from strain MC4100 (Casadaban 1976), partially digested with Sau3AI, and size fractionated by agarose gel electrophoresis. DNA fragments between approximately 1 kb and 4 kb in size were ligated with pBR322 DNA that had been digested with BamHI and dephosphorylated (Sambrook et al. 1989). The resultant library was transformed into D10 by electroporation, and transformants were selected on LB in the presence of ampicillin (125 mg/l) at 20°C. Although prolonged incubation led to high levels of background growth, cold-resistant colonies were easily distinguishable by their early appearance.

The cold-sensitive phenotype of strain D10 is suppressible by bipA

Approximately 12,000 transformants were analyzed for cold resistance and six such colonies were identified. These six colonies were purified and restreaked at 20°C to verify the cold-resistant phenotype. Plasmid DNA was purified from each isolate and retransformed into D10 to confirm that the cold resistance was plasmid encoded. All transformants displayed a cold-resistant phenotype, demonstrating that each contained a plasmid which suppressed the cold sensitivity of D10 (for example, pPLP7 in Fig. 2A).

Fig. 2A-C. Plasmid-borne bipA complements the cold-sensitive defect of D10 (Flower stock) and AF600. Plasmid pPLP7 was obtained in the screen for multicopy suppressors, and contains a 2300-bp fragment encoding bipA. A Strains were grown on LB agar with 125 mg/l ampicillin at 20°C for 4 days. B, C. Strains were grown in LB broth with 100 mg/l ampicillin at 20°C in the BioScreen as described in Fig. 1. Again, measurements were taken every 30 min, but only every 5-h increment is shown. Symbols: closed squares (B), MG1655; open squares (B), AF600; closed triangles (C), D10 (E. coli Genetic Stock Center); open triangles (C); D10 (Flower stock). All points connected by solid lines indicate strains that carry control plasmid pBR322, data for strains with pPLP7 are shown with dashed connecting lines

The six plasmids were analyzed by restriction enzyme digestion; the size of the inserts ranged from approximately 2300 bp to 8200 bp and each exhibited a different restriction fragment pattern, demonstrating that each plasmid was an independent isolate. DNA sequence analysis was performed using oligonucleotide primers that hybridized to the pBR322 DNA flanking the insert and directed DNA synthesis toward the chromosomal insert. The sequence data revealed that all six plasmids contained DNA from the same region of the chromosome, at about 87 min (Blattner et al. 1997). The minimal overlap between plasmids indicated that the region responsible for conferring cold resistance corresponded to the bipA gene (formerly yihK), which encodes a GTPase that can be phosphorylated on tyrosine (Farris et al. 1998). Indeed, the plasmid with the smallest insert contained very little DNA external to bipA (67 bp upstream and 455 bp downstream, with no other predicted ORFs), suggesting that this gene was sufficient to suppress the cold-sensitive phenotype of D10.

The cold-sensitive D10 phenotype is due to disruption of the bipA gene

There are two potential mechanisms by which bipA could function as a multicopy suppressor of D10 cold sensitivity. D10 may contain a mutation in another gene that confers cold sensitivity, in which case bipA functions as extragenic suppressor of that defect, or D10 may have a mutation in bipA itself, manifesting as a cold-sensitive defect, which is complemented by plasmid-borne bipA. To understand the cause of the cold-sensitive phenotype it is important to differentiate between these possibilities.

To this end, we sought to establish whether D10 contains a mutation in the chromosomal bipA gene. We attempted to amplify the bipA from D10 by PCR, in order to determine the DNA sequence of the gene. However, we were unable to obtain a PCR product when D10 DNA was used as the template, although DNA from every other strain we examined resulted in a PCR product of the expected size. The negative result of the PCR led us to speculate that the region of the chromosome containing bipA might have sustained an insertion, deletion or other rearrangement that resulted in disruption of the bipA gene. To test this hypothesis, we performed Southern analysis with DNA isolated from D10 as well as from MC4100. The plasmid we had isolated with the largest insert from the bipA region (8200 bp) was used as the probe (pPLP6). The chromosomal DNAs were digested with three different restriction enzymes in separate reactions (PstI, EcoRI, or BamHI), electrophoresed on a 1% agarose gel and subjected to Southern analysis (Sambrook et al. 1989). The results demonstrated that the physical structure of the DNA in this region differed in the two strains (Fig. 3). The hybridization pattern exhibited by MC4100 DNA corresponded to predictions based on the DNA sequence (Blattner et al. 1997). However, in all cases, the D10 chromosome appeared to contain additional DNA in this region, as the hybridizing band was larger than that from MC4100 (PstI) and/or there was an additional hybridizing band (EcoRI and BamHI). While we were unable to determine the exact nature of the structural differences, it was clear that the two strains were not the same. This result supports our hypothesis that an alteration in the chromosome of D10 in the region of 87 min has resulted in an inactive bipA gene.

Fig. 3. Southern analysis of D10 and MC4100. DNAs were digested with the indicated restriction enzymes. The positions of molecular weight markers is shown on the right. The DNA was probed with pPLP6, containing 8200 bp of chromosomal DNA from the bipA region of the chromosome

Two observations are noteworthy at this point. First, D10 has not been reported to be cold sensitive, nor to carry a bipA mutation (Gesteland 1966); second, a cold-sensitive phenotype has not been reported previously for cells bearing a bipA deletion (Farris et al. 1998). To determine whether the cold sensitivity is inherent to D10 or whether our strain had an additional mutation, we obtained D10 from the E. coli Genetic Stock Center (Yale University, New Haven, Conn.) and compared phenotypes. Unexpectedly, the D10 from the Genetic Stock Center was not cold sensitive (Fig. 1). In comparing the two strains further, we found that both exhibited the expected methionine auxotrophy (Gesteland 1966), but differed in their sensitivity to bacteriophage M13. While D10 was originally derived from an Hfr21 strain, it was noted in the original report of its isolation that D10 had apparently lost the ability to transfer DNA, as if it had lost the Hfr, yet it was still sensitive to male-specific phages (Gesteland 1966). Similarly, the E. coli Genetic Stock Center notes that D10 was Hfr21, but now appears to be F^-. We found that the D10 from the Genetic Stock Center was resistant to bacteriophage M13, while our D10 was sensitive to M13, like the original isolate. Thus, while there appeared to be slight differences in the genetic properties of the two strains, potentially related to the presence of an Hfr, our version of D10 was closely related to the originally described strain.

To verify that the cold-sensitive defect of our D10 was due to disruption of the bipA gene, we constructed a precise, complete deletion of bipA. Plasmid pAF68 was constructed using pTSC29 as the parent vector; pTSC29 encodes chloramphenicol resistance and constitutes a temperature-sensitive replicon (Phillips 1999). DNA that flanks bipA on the chromosome (1 kb upstream from bipA and 1.1 kb of downstream DNA) was inserted into pTSC29, with a kanamycin resistance cassette (from pCK155; Kristensen et al. 1995) placed between the upstream and downstream DNA. The resulting plasmid (pAF68) was transformed into strain MG1655 by selecting for kanamycin resistance at 42°C. After several cycles of growth at 30°C followed by growth at 42°C, resolved cointegrates that had lost the plasmid DNA but retained the kanamycin resistance cassette were identified by screening for chloramphenicol sensitivity and kanamycin resistance. These colonies represented mutants in which the bipA gene had been replaced with the kanamycin resistance cassette. This insertion-deletion mutation was verified by PCR amplification of the bipA region; those colonies that contained the kanamycin resistance cassette resulted in amplification of a larger fragment (2.2 kb) than those that retained bipA (1.9 kb).

The resultant bipA::kan strain (AF600) was assayed for its growth characteristics at 20°C. As expected, deletion of bipA resulted in a cold-sensitive phenotype, detectable both by lack of colony formation on LB agar plates and by a decreased growth rate in LB broth (Fig. 1). Furthermore, the cold sensitivity was complemented by plasmid pPLP7 encoding bipA (Fig. 2). These results confirmed that the cold sensitivity of D10 was due to lack of functional BipA, and, furthermore, complementation of the cold sensitivity by bipA demonstrated that the cold sensitivity was due to deletion of bipA and not to a polar effect on downstream genes. We note that both versions of D10 exhibit a slightly decreased rate of growth at 20°C relative to MG1655 (Fig. 1B). In addition, our D10 does not achieve the same final cell density as the D10 from the E. coli Genetic Stock Center, even at 37°C, although the rate of exponential growth appears similar (Fig. 1C). These results may indicate that further, uncharacterized defects are present in D10. Nevertheless, the growth differences between AF600 and its isogenic parent, MG1655, demonstrate that deletion of bipA results in a cold-sensitive growth defect that is rescued by plasmid-borne bipA.

The roles of BipA in pathogenesis and at low temperature

BipA was originally described as a protein that is induced in Salmonella typhimurium after exposure to bactericidal/permeability-inducing protein (a cationic antimicrobial protein produced by neutrophils) (Qi et al. 1995), and has since been identified in E. coli as well (Plunkett et al. 1993). The properties thus far described for BipA are related to its role in the process of pathogenesis by enteropathogenic E. coli; it appears to be involved in the regulation of several processes important for infection, including rearrangements of the cytoskeleton of the host, bacterial resistance to host defense peptides, flagellum-mediated cell motility (Farris et al. 1998), and expression of K5 capsular genes (Rowe et al. 2000). BipA belongs to the GTPase superfamily that includes elongation factors EF-G and EF-Tu, and the TetM/TetO tetracycline resistance proteins (Qi et al. 1995), and it has been proposed that BipA may utilize a novel mechanism to regulate the expression of target genes (Farris et al. 1998). In addition, BipA from enteropathogenic E. coli has been shown to be phosphorylated on a tyrosine residue, while BipA from Salmonella and from E. coli K12 strains is not phosphorylated under the conditions assayed (Farris et al. 1998). The phosphorylation apparently modifies the rate of nucleotide hydrolysis, with the phosphorylated form showing greatly increased GTPase activity.

Although the reported activities of BipA are related to pathogenesis by enteropathogenic E. coli, the results presented here demonstrate that BipA has an important and more general regulatory function in E. coli K12. It is likely that BipA activity is necessary for some aspect of E. coli physiology required at low temperatures, which is independent of pathogenicity. Furthermore, as BipA from E. coli K12 is not phosphorylated, it is apparent that phosphorylation is not required for this aspect of BipA function. Thus, the regulatory role of BipA is separable from the phosphorylation. Further investigation will be required to determine the role of BipA and the basis for the requirement for this protein at low temperatures.

Acknowledgements. We are grateful to Tom Henderson for assistance with the Southern blots, Kathy LaVoi for DNA sequencing, and Kevin Young for critical reading of the manuscript. This work was supported by the ND EPSCoR AURA program (PLP) and by CAREER Award MCB-96000851 from the National Science Foundation (AMF)

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