Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.

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Bacterial strain G n T (T = type strain), a nonmotile, nonsporeforming, mesophilic, methanogenic bacterium, was isolated from the primary sludge obtained from the waste treatment facility of a major kraft pulp mill in Canada. Single cells were 6.0 by 0.8 pm and stained gram positive. Growth and methane production occurred only with H,-CO, as the substrate. Acetate, formate, propionate, butyrate, pyruvate, methanol, or trimethylamine could not serve as a sole source of carbon and energy for growth. The optimum pH for growth was between 5.6 and 6.2; consistent growth and methane production were not observed below pH 4.68. The optimum temperature for growth was 35"C, and little or no growth was observed during incubation at 15 and 50°C. Kanamycin and bacitracin were severe inhibitors of growth and methanogenesis, whereas 100 pM bromoethanesulfonic acid caused 30 % inhibition. Supernatant from primary sludge enhanced growth by about 10%. The DNA base composition was 34 mol% guanine plus cytosine. On the basis of physiological characteristics, indirect immunofluorescence typing, and DNA-DNA hybridization studies, the isolate is named Methanobacterium espanolae sp. nov.The optimum pH for growth of most pure cultures of methanogenic bacteria isolated to date lies between 6.0 and 8.0, with the majority of cultures having an optimum close to pH 7.0 (23,25). Methanogenic bacteria with optimum growth pH values greater than 8.0 have been reported recently (27). Although methanogenic activity has been detected in acidic ecosystems, such as bogs and peats (11,24,25), at pH values close to 4.0, methanogenic isolates from these ecosystems have been unable to grow at pH values below 5.3 (25). Moreover, no pure culture of a methanogen that is able to grow at pH values below 5.0 has been reported previously, to our knowledge. The isolation and study of acid-tolerant methanogens was undertaken as part of a program on biomass degradation in anaerobic, acidic environments, with the purpose of extending the potential of anaerobic treatment for disposal of acidic, organic, industrial wastes. In this paper we describe the isolation and characterization of a methanogen that is able to grow and produce methane at pH values below 5.0. MATERIALS AND METHODSMedia. The composition of SA medium, which was used for isolation and stock culture maintenance, was as described previously (5) except for an increase in the cysteineNa,S concentration. This medium contained (per liter) 2,500 mg of CH,COONa -3H@, 480 mg of Na,CO,, 450 mg of mg of vitamin B12. The pH of the mixed ingredients (except cysteine-Na,S and Na,CO,) was adjusted to 4.5 with 5 N HC1. The medium was then reduced with cysteine-Na,S, Na,CO, was added, and 10-ml portions were dispensed into 60-ml serum vials under 80% H2-20% CO, (5). The postautoclaved (121"C, 15 min) pH of the medium was 5.5 k 0.1. Similarily, SA medium with an initial pH of 5.0 and S medium (the same as SA medium but without acetic acid) with pH values of 5.5 and 5.0 were prepared by appropriate pH adjustment before...

Autolvtic enzymes were found to be required for flagellai morphogenesis in Bacillus subtilis 168 and Bacillus lichen ifortinis 6346. Two previously characterized, poorly lytic, chain-fornming mutants of B. subtilis 168, strains FJ3 (temperature conditional) and FJ6, each 90 to 95%' deficient in the piroduction of Nacetylmuranml-L-alanine amidase and endo-/3-N-acetylglucosaminidase, were observed to be nonmotile at 35°C in a varietv of liquid and senmisolid media. In contrast, cells of the isogenic wild-type strain were motile and fully separated. Electron microscopy revealed the complete absence of flagella on the mutant cells. Similar observations were made with another poorly lytic strain of B. subtilis 168 (Nil5) and with two poorlv lytic, phosphoglucomutase-deficient mutants of B. licheniformis 6346 (MH-:3, MH-5). In minimal media lacking galactose (restrictive conditions), the B. lichen iformis mutants failed to form flagella, or had serious abnormalities in flagellar morphogenesis and motility. Under permissive conditions, mutants FJ3 (grown at 17°C) and MH-5 (grown with added galactose) showed increased autolytic activities, grew in the dechained form, and regained their capacities to synthesize functional flagella. Exanmination of several classes of spontaneous revertants derivred from the various mutant strains further demonstrated a close relationship) between autolvsin activity and flagellation in the two Bacillus spp).Autolysins are a group of cell wall-degrading enzymes which occuIr widely in bacteria (22, 38). These enzymes hydrolyze specific chemical bonds in the peptidoglNcan component of the procaryotic cell wall. They have a variety of specificities (38), and several have been p)urified to homogeneity (for examples, see ref. 21, 23). Under conditions of unbalanced growth (34), autolysins can bring about the dissolution of a bacterial cell, a phenomenon known as autolysis. The regulation of these potentially lethal bacterial enzymes has been the subject of recent investigations (10,21, 23).Vegetative cells of Bacillus subtilis 168 and 'his stUdyVI This study' Selected for motilitv on a semisolid medium (Pen A, NGA, or SGGA). Noninducible for defective phage PBSX (31).These phosphoglucomutase-deficient (Pgm ) strains are poorlI lytic. " Selected for ability to grow with galactose as a sole carboni soLlice. These P'gmstrains are phenotypicall L t>

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