In Klebsiella pneumoniae NCIB 418, the pathways normally responsible for aerobic growth on glycerol and sn-glycerol 3-phosphate (the glp system) are superrepressed. However, aerobic growth on glycerol can take place by the intervention of the NAD-linked glycerol dehydrogenase and the ATP-dependent dihydroxyacetone kinase of the dha system normally inducible only anaerobically by glycerol or dihydroxyacetone. Conclusive evidence that the dha system is responsible for both aerobic and anaerobic dissimilation of glycerol was provided by a Tn5 insertion mutant lacking dihydroxyacetone kinase. An enzymatically coupled assay specific for this enzyme was devised. Spontaneous reactivation of the glp system was achieved by selection for aerobic growth on sn-glycerol 3-phosphate or on limiting glycerol as the sole carbon and energy source. However, the expression of this system became constitutive. Aerobic operation of the glp system highly represses synthesis of the dha system enzymes by catabolite repression.

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA.The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and'has a pre-pro region of 60 amino acids. The mature B sublinit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa forn of inhibin also isolated from bovine follicular fluid, with subunits qf 20 kJa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.Considerable evidence has now accumulated to support the concept that the gonads produce a protein termed inhibin (1) that selectively suppresses the pituitary secretion of folliclestimulating hormone (2), a key hormone in controlling folliculogenesis and spermatogenesis. Controversy still exists concerning the nature of inhibin (3) partly because of the fact that some of the bioassays used in its characterization do not monitor follicle-stimulating hormone directly (4). For instance, inhibin-like activity has been detected in seminal plasma; the proteins associated with this activity have been purified and characterized but subsequently they were shown not to be of gonadal origin (5)(6)(7)(8)(9).We have isolated inhibin from a gonadal source, bovine follicular fluid, with an apparent molecular mass of 58 kDa, composed of two subunits now designated A and B, of 43 kDa and 15 kDa, respectively, linked together by disulfide bonds (10). Subsequently, 32-kDa inhibin was isolated by others from porcine follicular fluid, with two subunits of 18/20 kDa and 13/14 kDa (11,12). We have also shown that a 31-kDa form of bFF inhibin, composed of 20-kDa and 15-kDa subunits, is generated during a pH precipitation step in the purification procedure (13) but the precise relationship between the different forms has been unknown. However, the biological activity of the 31-kDa form is neutralized in vitro by an antiserum raised against the 58-kDa form (13), suggesting that the 31-kDa inhibin is a processed form of the 58-kDa inhibin.This article describes the amino acid sequences of the two subunits of the 58-kDa bovine inhibin as determined fromn cDNA sequencing and reports that the 20-kDa subunit of the 31-kDa inhibin is derived from the 43-kDa subunit of the 58-kDa inhibin, thus clarifying the relationship between these different forms.MATERIALS AND MIETIODS NH2-Terminal Amino Acid Sequencing of the 58-kDa Inhibin. The 58-kDa inhibin was isolated from bFF (10).Briefly, this involved a four-step' purjfication procedure: (i) gel permeation chromatography on Sephacryl 3200 in 0.05 M ammonium acetate, (it) gel permeation chromatography on Sephadex G100 in 4 M acetic acid, (iii) reversed-phase HPLC on an Ultrapore RPSC column-(Beckman) using a 0.1% trifluoroacetic acid-acetonitrile-H2O gradient, and (iv) prepara...

Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source.

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Hybridization histochemistry has been used to detect the presence of mRNA for the \ g=a\and \ g=b\ A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine \g=a\or \ g=b\ A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The \g=a\subunit mRNA was located in the granulosa layer of antral follicles >0\m=.\36mm in diameter while the \g=a\and \ g=b\ A subunit mRNA were both present in follicles of >0\m=.\8mm. In these latter follicles, the thecal layer hybridized with only the \ g=a\ subunit mRNA. No hybridization of the \ g=a\ or \ g=b\ A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the \g=a\inhibin subunit can be detected in granulosa and theca cells whereas the \ g=b\ A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.

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Entomopathogenic bacteria of the genus Xenorhabdus produce crystalline inclusion bodies during in vitro culture. When cultured in liquid media, inclusions were present in primary forms but not secondary forms of X. nematophilus. In contrast, both primary and secondary forms of X. luminescens produced inclusion bodies during liquid culture. Two morphologically distinct forms of inclusion bodies were found in X. nematophilus subsp. nematophilus strain All. They were proteinaceous, and one of the proteins (IP-1) was present in seven strains of X. nematophihrs subsp. nematophilus, but not in strains of X. nematophilus subsp. bovienii, X. nematophilus subsp. poinarii, X. nematophilus subsp. beddingii or X. luminescens. This protein may be useful as a taxonomic indicator. Plasmids were isolated from seven of ten strains of Xenorhabdus. They varied in size from 12 to 3.6 kb, and were present in both primary and secondary forms of X. nematophilus subsp. nematophilus and X. nematophilus subsp. bovienii.

Immunization of ewes against a pure recombinant preparation of the alpha subunit of bovine inhibin (alpha-bI) resulted in a three- to fourfold increase in ovulation rate, associated with antibodies in plasma recognizing pure native 31 kDa inhibin. The aim of this study was to examine the effects of this immunization on basal and GnRH-stimulated plasma concentrations of FSH and LH in ewes during the anoestrous and breeding seasons. The groups were untreated control ewes (n = 5), control ewes treated with keyhole limpet haemocyanin (KLH alone, n = 4), ewes treated with alpha-bI alone (n = 4) and alpha-bI-KLH conjugate-treated ewes (n = 3). There were no effects of immunization on basal FSH or LH in anoestrous ewes, despite the presence of antibodies recognizing 31 kDa inhibin. In the breeding season, immunization against alpha-bI resulted in increased basal (follicular phase, P less than 0.1; luteal phase P less than 0.05) and GnRH-stimulated (follicular phase only, P less than 0.001) release of FSH, but not LH. The data are compatible with the hypotheses that the increase in ovulation rate in immunized ewes is due to an increase in circulating FSH concentrations and that inhibin may only have a major peripheral influence on FSH in sheep during the breeding season.