SummaryThe suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42ЊC. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with ␤-lactam antibiotics. The ␤-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed ␤-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by ␤-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to ␤-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. KTG). Toho-1 was about 83% homologous to the ␤-lactamase mediated by the chromosome of K. oxytoca D488 and the ␤-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated ␤-lactamase Toho-1 produced by E. coli TUH12191 is similar to ␤-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A ␤-lactamase. Detailed comparison of Toho-1 with other ␤-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.Expanded-spectrum cephalosporins have chemical structures which confer stability to many ␤-lactamases from gram-negative bacteria. However, many members of the family Enterobacteriaceae other than Escherichia coli developed resistance to the expanded-spectrum cephems (40). The primary mechanism of this resistance was demonstrated to be excessive production of a chromosomal ␤-lactamase (AmpC) (23). However, bacteria that show resistance mediated by other ␤-lactamases appeared in 1984 (8). Species of the Enterobacteriaceae such as Klebsiella pneumoniae, Klebsiella oxytoca, and E. coli acquired resistance against expanded-spectrum cephem antibiotics by producing extended-spectrum ␤-lactamase. The extended spectrum of the ␤-lactamase was often acquired by the variation of ␤-lactamase genes on transmissible plasmids (43,44). Under the influence of antimicrobial agents, bacteria producing primarily TEM-type or SHV-type ␤-lactamases developed point mutations in structural genes which served to extend the substrate specificity of the enzymes (44). These TEM-type and SHV-type ␤-lactamases show about 65% amino acid sequence homology, with isoelectric points of 5.5 to 6.3 and 7.0 to 8.2, respectively (8, 9).

Bacterial resistance to ␤-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of ␤-lactamases that inactivate ␤-lactam antibiotics. Toho-1 is an extended-spectrum ␤-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former ␤-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-Å resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the ⍀-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn 104 and Asp 240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser 237 and cephalosporins probably helps to bring the ␤-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

We study nonlinear diffusion problems of the form ut = uxx + f (u) with free boundaries. Such problems may be used to describe the spreading of a biological or chemical species, with the free boundaries representing the expanding fronts. For monostable, bistable, and combustion types of nonlinearities, Du and Lou ["Spreading and vanishing in nonlinear diffusion problems with free boundaries," J. Eur. Math. Soc. (JEMS), to appear] obtained a rather complete description of the long-time dynamical behavior of the problem and revealed sharp transition phenomena between spreading (limt→∞ u(t, x) = 1) and vanishing (limt→∞ u(t, x) = 0). They also determined the asymptotic spreading speed of the fronts by making use of semiwaves when spreading happens. In this paper, we give a much sharper estimate for the spreading speed of the fronts than that in the above-mentioned work of Du and Lou, and we describe how the solution approaches the semiwave when spreading happens.

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