- Volume 143, Issue 10, 1997

Sera from 10 patients infected with Mycoplasma pneumoniae were used in Western blot analysis of Triton-X-114-soluble protein preparations of M. pneumoniae. All 10 sera were reactive with a protein antigen of 116 kDa. Sera from another 17 patients were used in Western blot analysis of whole-cell M. pneumoniae proteins; 15 of these sera were reactive with the 116 kDa protein. Trypsin digestion of whole M. pneumoniae cells demonstrated the surface location of this protein. Sequencing of DNA which contained the gene for this protein identified an ORF of 3093 bp encoding a protein with a predicted molecular mass of 116013 Da. The ORF for the 116 kDa protein had 99·8% nucleotide identity with the M. pneumoniae gene G07_orf1030 and 61% nucleotide identity with the Mycoplasma genitalium ORF MG075 of unassigned function. An ORF which was identified 5' to the 116 kDa protein ORF coded for a 16 kDa protein and had 99·8% nucleotide identity with the M. pneumoniae gene G07_orf135 and 58·4% nucleotide identity with the ORF MG074 of M. genitalium. Analysis of mRNA detected a 3·7 kb transcript with a single initiation site 5' to the ORF encoding the 16 kDa protein. The coding sequences for both the 16 kDa protein and the 116 kDa protein were present in this transcript, indicating that they were part of an operon and suggesting a possible functional relationship.

During a search for genes encoding electron transport proteins from a Thiobacillus ferrooxidans ATCC 33020 gene bank, a 19.8 kb plasmid, pTF5, which conferred increased sensitivity to the antimicrobial agent metronidazole upon an Escherichia coli mutant, was isolated and cloned in E. coli. The plasmid had an identical restriction enzyme map to a plasmid which has been found in T. ferrooxidans strains isolated from many different parts of the world. The plasmid was present at between two and four copies per genome and contained a region of approximately 5.6 kb which was also found on the chromosome. This region was sequenced and found to have four complete ORFs, which when translated had high percentage amino acid similarity to [3Fe-4S,4Fe-4S] ferredoxins, proteins of the FNR regulator family, prismane-like proteins and the NADH oxidoreductase subunit of a methane monooxygenase. In vitro protein analysis using an E. coli-derived transcription-translation system indicated that three of the four products (FdxA, PsmA and RedA) were expressed in the heterologous system. Ferredoxins, prismane-like proteins and NADH oxidoreductases are redox-active proteins and it is likely that the proteins on pTF5 represent an electron transport system of as yet unknown function. Surprisingly, although genes for redox-active proteins have been isolated from other bacteria by screening gene banks for increased sensitivity to metronidazole, the region of pTF5 containing the genes for these proteins was not responsible for the increase in metronidazole sensitivity conferred by the plasmid. The region of pTF5 which did confer increased metronidazole sensitivity to an E. coli metronidazole-resistant mutant was a 319 bp region of DNA close to the origin of plasmid replication. This region contained no ORFs and was identical to that previously reported for the replicon of a 9.8 kb T. ferrooxidans plasmid, pTFI91.

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

A glyceraldehyde-3-phosphate dehydrogenase (gpd) cDNA was isolated from the filamentous fungus Trichoderma harzianum in the course of a search for light-regulated genes in this organism. There is apparently only one copy of gpd in the T. harzianum genome, and its sequence is most similar to that of other filamentous ascomycetes. Trichoderma grows in the soil as a saprophyte or mycoparasite. A brief pulse of blue light, or nutrient depletion, induces sporulation, which is accompanied by altered patterns of abundance of specific polypeptides. Mycoparasitic development is also accompanied by changes in gene expression. The abundance of gpd mRNA decreased strongly during sporulation, and was lowest in samples consisting of mature conidiophores and conidia. When T. harzianum was grown in the presence of cell walls of the phytopathogen Rhizoctonia solani, the gpd mRNA level was much lower than in similar cultures grown on glucose. The repression of gpd, which is usually considered a constitutively expressed gene, may be part of the switch to sporulation or to the simulated mycoparasitic state. The implications of these findings for the use of gpd promoters to confer high constitutive expression are discussed.

Colicins are divided into two groups according to the proteins required for their import into sensitive bacteria. The Tol and TonB pathways are involved in import of group A and group B colicins respectively. Because previous analyses have shown that colicin E1 and colicin A (two group A colicins) interact in vitro with the C-terminal domain of TolA (TolAIII) while colicin B (group B colicin) does not, attention was focused on these interactions with purified proteins. TolA has been described as a three-domain protein with an N-terminal inner-membrane anchor and a long periplasmic region formed by two domains (TolAII and TolAIII). TolAIII, TolAII and TolAII-III soluble domains with an N-terminal hexa-histidine extension were purified. The interactions of colicins with the purified TolA domains were analysed by overlay Western blotting, which indicated that both N-terminal domains of colicins A and E1 interacted with TolAIII, while a gel shift procedure detected no interaction with colicin E1. The binding kinetic values of the N-terminal domains of colicins A and E1 to TolAIII were estimated by surface plasmon resonance and were shown to be similar.

Two chemotactic transducer genes (termed pctB and pctC and an open reading frame (orf1) were found in the pctA-flanking region which was previously identified as a chemotactic transducer gene in Pseudomonas aeruginosa. The pctB and pctC genes encode predicted polypeptides of 629 and 632 amino acids, respectively. Overall, PctB and PctC had 81 and 75% amino acid identities with PctA, respectively. A null mutant strain PCT2, which contained a deletion in the entire pcfC, orf1, pctA and pctB genes, did not show chemotaxis towards all 20 commonly occurring L-amino acids. This mutant strain also failed to respond to amino acid catabolites (cadaverine, 4-aminobutyrate and putrescine) that are strong attractants for the wild-type strain PAO1. To study the role of each gene product in L-amino acid taxis, plasmids harbouring the pctC, orf1, pctA, or pctB genes were constructed and introduced into strain PCT2 by transformation. The orf1 gene did not complement the defect in chemotaxis of strain PCT2. The pctA gene restored the ability of strain PCT2 to respond to 18 L-amino acids, suggesting that PctA plays a major role in detecting L-amino acids in P. aeruginosa. The pctB and pctC genes complemented the defect in chemotaxis to only seven (Ala, Arg, Glu, Lys, Met, Tyr, Gin) and two (His, Pro) L-amino acids, respectively.

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the β D form of RNA polymerase. The level of β-gaiactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No β-gaiactosidase could be detected in a sigD genetic background.

High-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possibly via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.

The nucleotide sequence on both sides of the eryA polyketide synthase gene of the erythromycin-producing bacterium Saccharopolyspora erythraea reve the presence of ten genes that are involved in L-mycarose (eryB) and D-desosamine (eryC) biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryBIV, eryBV an eryBVI) act in L-mycairose biosynthesis or attachment, while the other five (eryCII, eryCIII, eryCIV, eryCV and eryCVI) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryBII and eryBVII) appear to function in L-mycarose biosynthesis based on computer analysis an earlier genetic data. Three of these genes, eryBII, eryCIII and eryCII, lie between the eryAIII and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryC and eryBVII lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexos 4-ketoreductases (eryBIV), aldoketo reductases (eryBII), aldohexose 5-epimerases (eryBVII), the dnmT gene of the daunomycin biosynthetic pathwa of Streptomyces peucetius (eryBVI), glycosyltransferases (eryBV and eryCIII), the AscC 3,4-dehydratase from the ascarylose biosynthetic pathway of Yersin pseudotuberculosis (eryCIV), and mammalian N-methyltransferases (eryCVI). The eryCII gene resembles a cytochrome P450, but lacks the conserved cysteir residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.

Alkaline extracellular protease (AEP) from Yarrowia lipolytica is synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Ala15 by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro17 to Met which should prevent DPAPase processing and Ala19 to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor Pro-boroPro. Deletion of the entire dipeptide stretch (Ala16 to Pro33) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type and dpp1 dap2 Saccharomyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that the KEX2-like cleavage after Lys156-Arg157, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.

Prototrophic revertants of a metH2 strain of Aspergillus nidulans which is impaired in the regulation of synthesis of folate-dependent enzymes were isolated and six of them analysed. In three of the isolates reversion was the result of an intragenic suppressor mutation in the metH locus. In the remaining strains suppressor mutations occurred in independent genes. These genes, designated folA, folB and folC, are linked and located in chromosome VI. Mutations in these genes render synthesis of some folate enzymes, particularly folylpolyglutamate synthetase, insensitive to methionine-mediated repression.

An Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1·8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequence identity was apparent with the 4-α-glucanotransferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated malQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MalP). The translation stop codon of malQ overlapped the translation start codon of the putative malP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-α-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MalQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.

The Bacillus subtilis α-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic context, α-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (t 1/2 = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of α-amylase occurring within the cell wall environment was substantiated by in vitro folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (t 1/2 = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wail. The t 1/2 of the α-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).

The antituberculosis drug pyrazinamide (PZA) needs to be converted into pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) in order to show bactericidal activity against Mycobacterium tuberculosis. M. avium is naturally resistant to PZA. To investigate whether this natural resistance to PZA is due to inability of the M. avium PZase to convert PZA to bactericidal POA, the M. avium PZase gene (pncA) was cloned by using the M. tuberculosis pncA gene as a probe. Sequence analysis showed that the M. avium pncA gene is 561 bp long, encoding a protein with a predicted size of about 19·8 kDa; but Western blotting showed that the M. avium PZase migrated as a 24 kDa band when expressed in M. bovis BCG and Escherichia coli. Sequence comparison revealed that M. avium PZase has 67·7% and 32·8% amino acid identity with the corresponding enzymes from M. tuberculosis and E. coli, respectively. Southern blot analysis with the M. avium pncA gene as a probe showed that M. terrae, M. gastri, M. marinum, M. fortuitum, M. xenopi, M. gordonae, M. szulgai, M. celatum and M. kansasii have close pncA homologues, whereas M. chelonae and M. smegmatis did not give significant hybridization signals. Transformation with the M. avium pncA gene conferred PZA susceptibility to PZA-resistant M. tuberculosis complex organisms, indicating that the nonsusceptibility of M. avium to PZA is not due to an ineffective PZase enzyme, but appears to be related to other factors such as transport of POA.

An improved integrative cassette from plasmid pSAM2 has been constructed containing plasmid int and attP genes but excluding the xis gene, which should result in increased stability by suppression of the excision reaction. This cassette was included in both suicide and thermosensitive plasmids and used for integration in Mycobacterium smegmatis. Suicide plasmids containing this cassette integrated at a single site (attB1) in the M. smegmatis chromosome. The sequence of the attB1 site has been determined and was identified as a putative tRNAPro gene. Thermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to simultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.