- Volume 131, Issue 10, 1985

Summary: Studies of the hydroxylated fatty acids in strains of Nocardia otitidis-caviarum showed variations in mycolic acid patterns, some strains containing only nocardomycolic acids and other strains having both nocardomycolic and corynomycolic acids. Mass spectroscopy showed that most of the nocardomycolic acids contained 52 to 56 carbon atoms and two double bonds in the main chain; the corynomycolic acids had saturated or monounsaturated chains and 30 to 34 carbon atoms. In the strains having both types of mycolic acid, only nocardomycolic acids were found as cell wall components.

Summary: Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 °C, were isolated. One ts plasmid was unstable at 42 °C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.

Summary: The phylogenetic relationship of the Gram-negative, filamentous gliding bacterium Filibacter limicola was analysed by 16S rRNA oligonucleotide cataloguing. In contrast to the proposed membership of this asporogenous species in the Flexibacteriaceae, Filibacter limicola clusters phylogenetically with the Gram-positive eubacteria Bacillus pasteurii, Sporosarcina ureae and the asporogenous species Planococcus citreus. The genetic relationship is supported by several common phenotypic properties.

Summary: Previous work has shown that Escherichia coli K 12 ColE2+ cells undergo a form of partial lysisand exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficientcolicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPEcontent. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K 12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K 12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E.coli K 12 as the model strain for studying the mechanisms of colicin release.

Summary: Regulation of NAD biosynthesis was examined through the construction of nad–lac fusions in Salmonella typhimurium. The nad A (17 unit map position) and nadB (55 units) genetic loci involved with quinolinic acid biosynthesis were both found to be regulated by the product of a nadR locus (99 units) in a repression/derepression manner while nadC (3 units) expression appeared constitutive at the transcriptional level. Increases in nadAB transcription directly correlated with decreases in intracellular NAD(P) levels, and kinetic studies indicated that the NAD analogue 6-aminoNAD was ineffective in repressing either nadA or nadB. The presence of cAMP + cAMP receptor protein was essential for the complete derepression of nadA while no effect was evident upon nadB. Transfer of cultures from aerobic to anaerobic conditions, however, resulted in the partial derepression of both nadA and nadB. Thus, there appears to be a very complex set of controls regulating NAD biosynthesis.

Summary: Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities. These NirB−strains synthesize cytochrome c 552 and grow normally on anaerobic glyeerol–fumarate plates. The defects map in a gene, nir B, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB. Complementation studies established that nirB + and cysG + canbe expressed independently. The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4).

The nirB gene is apparently defective in the previously described nirD mutant, LCB82. The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c 552 was detected. Strains carrying a nirE, nirF or nirG mutation gavenormal rates of nitrite reduction by glucose, formate or NADH.

Summary: Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain 834. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucoseon sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated.

Summary: Thermophilic mutants were isolated from mesophilic Bacillus suhtilis and Bacillus pumilus by platinglarge numbers of cells and incubating them for several days at a temperature about 10 °C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 °C and 70 °C at a frequency of less than 10−10. The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10−3 to 10−2 for single markers and about 10−7 for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10−7, suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.

Summary: In several strains of Bacillus subtilis extensive breakdown of chromosomal DNA may be potentiated by osmotic lysisof protoplasts. At its most severe, in strains originating from Farmer& Rothman’s thymine auxotroph, the rate of DNA breakdown was greater than 50% per hour at 40 °C. The rate of DNA breakdown in most other strains tested was approximately 5% per hour except for SP β-strains, in which the rate of DNA breakdown was only 0.3 %. DNA degradation was attributed to relaxation ofcontrol of a nuclease specified by the prophage of SP β or a related phage. The most potent nuclease in lysates was an ATP-activated protein of M r 280000. Derivatives of Farmer and Rothman’s strain containing integrated plasmids had the highest rate of DNA degradation. Although the chromosome was completely destroyed, covalently closed circular plasmids were generated from the integrated sequence. These showed massive deletions of the B. subtilis part of the integrated plasmid but the vector sequence remained intact. The nucleolytic activity therefore appears to recognize specific sequences in B. subtilis DNA. We suggest that activation of SP β genes during development of competence may be a cause of deletion ofcloned genes in the early stages of establishment of cloned sequences.

Summary: At least one plasmid from each of the incompatibility groups B, C, FIV, H2/S, Iα, Iδ, P, W and X was shown to be capable of transfer from Escherichia coli K12 to Acinetobacter calcoaceticus EBF65/65. Transfer was influenced by the presence of pAV2 (thought to encode a restriction-modification system) in the recipient strain; however, not all plasmids belonging to a particular incompatibility group behaved identically. All plasmids were unstable to varying degrees in A. calcoaceticus EBF65/65, but under suitable conditions were capable of transfer to further strains of EBF65/65 and re-transfer to E. coli K12. Of 40 recently isolated trimethoprim R plasmids 31 transferred successfully from E. coli K12 to A. calcoaceticus EBF65/65, but 17 of these 31 required the introduction of a second mobilizing plasmid for re-transfer to occur.

summary: Protoplasts of nutritionally complementary strains of Cephalosporium acremonium were fused and plated onto media which supressed the growth of both parents. The regenerating colonies were used for genetic analysis and were found to be of two types, stable haploid recombinants and unstable heterozygotes (aneuploids and/or diploids). Analysis of these colonies provided evidence for eight linkage groups and a relatively high rate of mitotic crossing-over. The gene order for three of the markers on one linkage group was also determined.

Summary: A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135000 vs 145000, as estimated by SDS polyacrylamide gel electrophoresis).

Summary: A λ 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in legnth. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.

Summary: Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.