- Volume 142, Issue 7, 1996

The Hha protein from Escherichia coli is highly similar (82%) to the YmoA protein from Yersinia enterocolitica. Both are members of a new class of proteins that modulates gene expression, probably by influencing DNA topology. In this paper, complementation of the hha mutation in E. coli by the ymoA gene from Y. enterocolitica has been studied. We show that the ymoA gene complements one of the phenotypic properties of hha mutants (high level of haemolysin production when they carry the recombinant plasmid pANN202-312) when cloned in a medium-copy-number plasmid but not when carried in a low-copy-number plasmid. Western blot analysis of the expression of YmoA in E. coli rules out inefficient expression of the protein. Surprisingly, the hha gene itself fails to complement the hha mutation when cloned in a medium-copy-number vector and causes genetic rearrangements of the E. coli chromosome as a consequence of insertion sequences mobilization.

A previously described DNA probe specific for Mycoplasma hyopneumoniae (l-141) was fully sequenced and found to consist of 1618 bp and to contain two tandemly repeated ORFs. The deduced amino acid sequence of the two ORFs showed significant homologies with ATP-binding cassette (ABC) transporter proteins, particularly those of the eukaryotic multidrug resistance (MDR) protein family (up to 21 % identity and 47% similarity). A somewhat lower homology was evident with the secretion protein HlyB of the RTX-haemolysin from Escherichia coli. The location of the two ORFs on the M. hyopneumoniae chromosome was downstream of the rrl gene encoding the 23S rRNA, but transcribed in the opposite direction. PCR amplification and subsequent chromosomal analysis by Southern blot hybridization of several M. hyopneumoniae strains showed that all field strains contained the two putative ABC transporter genes. However, some culture collection strains derived from strain J had lost these genes as the result of a 2221 bp deletion.

An acid-sensitive mutant TG5-46, derived from Rhizobium meliloti WSM419 by Tn5 mutagenesis, fails to grow below pH 6.0 whereas the parent strain grows at pH 5.7. The DNA sequence of a 2.2 kb rhizobial DNA region flanking Tn5 in TG5-46 contains two open reading frames, ORF1 (designated actS) and ORF2 (designated actR), having high similarity to the sensor-regulator pairs of the two-component systems involved in signal transduction in prokaryotes. Insertion of an omega interposon into actS in R. meliloti WSM419 resulted in an acid-sensitive phenotype. A DNA fragment from the wild-type complemented the acid-sensitive phenotype of RT295 (ActS-) and TG5-46 (ActR-), while fragments containing only actR or actS complemented TG5-46 and RT295, respectively. The presence of multiple copies of actR complementes not only TG5-46 but also RT295. Cloning DNA upstream from actR and actS into a broad-host-range lacZ expression vector and measuring β-galactosidase activities showed that both genes are constitutively expressed regardless of the external pH. Genomic DNA from all strains of R. meliloti, but no other bacteria tested, hybridized with an actRS probe at high stringency. These data implicate a two-component sensor-regulator protein pair in acid tolerance in R. meliloti and suggest their involvement in pH sensing and/or response by these bacteria.

An active-transport system for the osmoprotective compound glucosylglycerol (GG) was found in the cyanobacterium Synechocystis sp. strain PCC 6803. Uptake assays with 14C-labelled GG showed that the GG transport was enhanced in cells adapted to increasing concentrations of NaCl. Kinetic studies indicated a Michaelis-Menten relationship. The uptake of GG was energy dependent and occurred against a steep concentration gradient. It was inhibited by uncouplers as well as by a combination of darkness and KCN. The affinity of the transporter seems to be restricted to osmoprotective compounds of cyanobacteria; from a variety of compounds tested only sucrose and trehalose competed with GG for uptake. A salt-sensitive mutant of Synechocystis 6803 unable to synthesize GG could be complemented to salt resistance by exogenous GG. Accumulation of GG from the medium was essential for the restoration of photosynthesis and growth in mutant cells under high-salt conditions. In wild-type cells, the GG transporter probably serves to prevent GG leaking out of salt-stressed cells.

The essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.

Addition of glucose or fructose to cells of the yeast Saccharomyces cerevisiae grown on a nonfermentable carbon source triggers within a few minutes post-translational activation of trehalase, repression of the CTT1 (catalase) and SSA3 (Hsp70) genes, and induction of the ribosomal protein genes RPL1, RPL25 and RPS33. By using appropriate sugar kinase mutants, it was shown that rapid glucose- or fructose-induced activation of trehalase requires phosphorylation of the sugar. On the other hand, partial induction of RPL1, RPL25 and RPS33 as well as partial repression of CTT1 and SSA3 were observed in the absence of sugar phosphorylation. In glucose-grown nitrogen-starved yeast cells re-addition of a nitrogen source triggers activation of trehalase in a glucose- or fructose-dependent way, but with no apparent requirement for phosphorylation of the sugar. Repression of CTT1 and SSA3 under the same conditions was also largely dependent on the presence of the sugar and also in these cases there was a strong effect when the sugar could not be phosphorylated. Nitrogen induction of RPL1, RPL25 and RPS33 was much less dependent on the presence of the sugar, and only phosphorylated sugar caused a further increase in expression. These results show that two glucose-dependent signalling pathways, which can be distinguished on the basis of their requirement for glucose phosphorylation, appear to be involved in activation of trehalase, repression of CTT1 and SSA3 and induction of ribosomal protein genes. They also show that nutrient-induced repression of CTT1 and SSA3 is not a response to improvement of the growth conditions because the addition of nonmetabolizable sugar does not ameliorate the growth conditions. Similarly, the upshift in ribosomal protein synthesis cannot be a response to increased availability of energy or biosynthetic capacity derived from glucose, but it is apparently triggered to a significant extent by specific detection of glucose as such.

When Saccharopolyspora erythraea biomass from submerged culture was filtered (100-120 μn sintered glass filter) the antibiotic yield of the retentate (mean hyphal particle diameter 103 μm) was significantly higher [1.740 mg (mg biomass)-1] than that of the filtrate [1.286 mg (mg biomass)-1; mean hyphal particle diameter 88 μn]. A hypothesis to explain this is that there is a critical hyphal particle diameter, below which the particle is incapable of producing antibiotic. This would be a consequence of the site of antibiotic production occurring at a fixed distance from the growing hyphal tip. A protocol is proposed for calculation of the hypothetical critical hyphal diameter (88 μm in this case). The proportion of retentate (more productive) fraction of biomass varied between 60% and 30% during the course of a batch culture. Bioreactor stirrer speed significantly affected mean hyphal particle diameter (70 fim at 1500 r.p.m.; 124 μm at 750 r.p.m.) and antibiotic productivity [0.867 mg (mg biomass)-1 at 1500 r.p.m.; 0.913 mg (mg biomass)-1 at 750 r.p.m.].

Streptomyces clavuligerus produced cephamycin C under all nutrient limitations investigated in batch culture, whilst clavulanic acid was produced under phosphate and carbon limitations. Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) was produced at very low levels in all fermentations, prior to the detection of cephamycin C in the fermentation broth. Guanosine 5′-diphosphate 3′-monophosphate (ppGp) was observed at high levels in all fermentations; it appeared following a downturn in nutrient levels and immediately prior to detection of isopenicillin N synthase (IPNS). ppGp was detected following nutritional shiftdown by amino acid depletion and it was not produced via degradation of ppGpp. The results point to a possible role for ppGp in the regulation of cephamycin production in S. clavuligerus.

Streptomyces exfoliatus SMF13 sequentially produced leupeptin, leupeptin-inactivating enzyme (LIE) and trypsin-like protease (TLP). TLP was produced upon exhaustion of glucose. Autolysis of mycelium was accompanied by an increase in TLP activity. However, in three bld mutants isolated from S. exfoliatus SMF13 after UV-mutagenesis, mycelium autolysis did not occur, and neither LIE nor TLP was produced, although leupeptin was produced. Production of both LIE and TLP was restored in a spontaneous Spo+ revertant of a bld mutant. In contrast, two whi mutants sequentially produced leupeptin, LIE and TLP. The molecular mass of TLP produced during morphological differentiation was estimated to be 31.8 kDa by SOS-PAGE. The N-terminal amino acid sequence was RVGGTxAAQGNFPFQQxLSM. TLP was competitively inhibited by leupeptin; the inhibition constant was 0.015 μM. TLP effectively hydrolysed the mycelial protein extract of S. exfoliatus SMF13, but the hydrolytic activity was inhibited by leupeptin. It was concluded that morphological differentiation and production of TLP are coordinately regulated, that TLP may function as an enzyme in the metabolism of mycelial proteins, and that the hydrolytic activity of TLP is regulated by autogenous leupeptin in S. exfoliatus SMF13.

The growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated. In batch culture, growth was exponential and growth rate (μ) and yields (Y) were high [μ = 0.51 h-1 and Y X/benzoate = 0.56 mol carbon (mol carbon)-1] when low concentrations of benzoate (< 5 mM) were used. These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat [μmax = 0.55 h-1 and Y max X/benzoate = 0.57 mol carbon (mol carbon)-1] and the part of the energy for maintenance was limited (m ATP = 4.3 ± 2.2 mmol ATP g-1 h-1). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. The specific rate of benzoate consumption was not altered, whereas growth was inhibited [Ki (benzoate) # 27 mM]. Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5-2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD- intracellular content. The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism. Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.

Intra-specific diversity within Pasteurella haemolytica was assessed by analysing variation in the capsular polysaccharide (serotypes), lipopolysaccharide (LPS) and outer-membrane proteins (OMPs) of 184 isolates recovered from cattle and sheep. Four of 12 serotypes comprised 83% of the total number of isolates, including A1 and A2 as the most frequently recovered serotypes from cattle and sheep, respectively. Nine distinct LPS profiles were identified. Four different core-oligosaccharide patterns were present, each of which occurred alone as rough LPS and also in association with a single O-antigen profile as smooth LPS; the ninth LPS type was also smooth but had a different O-antigen profile. The capsular serotypes could be divided into four groups based on the dominant LPS profile within each serotype: (1) A1, A6, A9, A12 and A5; (2) A2, A8, A14 and A16; (3) A7 and A13; and (4) A11. Smooth LPS of type 1A, which was found only in the first group, was associated primarily with bovine disease isolates, whereas rough LPS of types 1B and 3B were associated primarily with the group 2 serotypes and ovine disease isolates. Similarly, the variation of OMP profiles generated three groups: (1) A1, A6, A9, A12, A5 and A8; (2) A2, A14 and A16; and (3) A7, A11 and A13. Isolates belonging to groups 2 and 3 exhibited greater diversity in their OMP profiles than those belonging to group 1. Although the majority of group 3 isolates possessed profiles unique to that group, a smaller number of A7 isolates possessed profiles with similarities to those of serotypes A1 or A2. OMP profiles clearly differentiated bovine from ovine isolates of the same serotypes. The association both of specific LPS and OMP profiles with bovine or ovine disease isolates suggested a correlation between specific cell-surface structures and host specificity. The combined analysis of capsular serotypes, LPS types and OMP profiles identified seven major groups within P. haemolytica which were responsible for 59% of the disease cases, suggesting a clonal structure for this species. Overall, comparison of the capsular serotypes, LPS types and OMP profiles proved extremely useful for assessing diversity within P. haemolytica.

The lepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes lepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively. The lepA operon is located immediately upstream of the dnaK operon. Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure. The lepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between lepA and hemN. Northern blot experiments revealed only a transcript corresponding to lepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against Histagged HemN. The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator. Readthrough transcription into the downstream dnaK operon was not found.

A new Escherichia coli gene, bgIX, encoding a β-D-glucosidase (EC 3.2.1.21) has been characterized. The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E. coli chromosome. The sequence of a 2.6 kb DNA fragment from this region revealed a large open reading frame encoding a protein of 765 amino acids. The BgIX protein was purified from the peripiasm, and amino-terminal sequencing suggests that the mature protein is derived from cleavage of a 20 residue signal peptide. A search of the sequence databases revealed that BgIX is a member of a large family of β-glucosidases from a variety of bacteria and fungi, and the plant Arabidopsis thaliana. It differs from the other four E. coli phospho-β-glucosidases in sequence and in its periplasmic rather than cytoplasmic location. The BgIX enzyme has a K m of 18 ± 1 mM and a V max of 3 ± 0.7 μmol min-1 for the colorimetric substrate o-nitrophenyl β-D-glucopyranoside.