- Volume 138, Issue 7, 1992

Acinetobacter calcoaceticus ATCC 23055 produces a large amount of 1,3-diaminopropane under normal growth conditions. The enzyme responsible, L-2,4-diaminobutyrate (DABA) decarboxylase (EC 4.1.1.-), was purified to electrophoretic homogeneity from this bacterium. The native enzyme had an M r of approximately 108000, with a pl of 5.0, and was a dimer composed of identical or nearly identical subunits with apparent M r 53000. The enzyme showed hyperbolic kinetics with a K m of 1.59 mM for DABA and 14.6 μM for pyridoxal 5'-phosphate as a coenzyme. The pH optimum was in the range 8.5–8.75, and Ca2+gave a much higher enzyme activity than Mg2+as a cationic cofactor. N-γ-AcetyIDABA, 2,3-diaminopropionic acid, ornithine and lysine were inert as substrates. The enzyme was different in subunit structure, N-terminal amino acid sequence and immunoreactivity from the DABA decarboxylase of Vibrio alginolyticus previously described.

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyrA. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyrA gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyrA*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ α-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the K m of the TyrA* dehydrogenase for NAD+remained unaltered, the K m for prephenate was fourfold greater and the V max was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyrA* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the K i value for L-tyrosine was decreased from 66 μM to 14 μM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein. These include alterations in apparent substrate specificity, isoelectric point, stability, catalytic properties and regulatory properties.

Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G + C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.

The structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118. Nucleotide sequence analysis revealed a gene of 936 bp. lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids. The mature protein was predicted to have a M r of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases. Furthermore it is highly homologous (>60%) to other lipases from various pseudomonads. The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P. alcaligenes, even under relaxed stringency. Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M r 33587) that shows some homology to the limA gene product of P. cepacia. In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P. aeruginosa PAO1.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd−) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(ohydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd−mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd−mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd−, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd−phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmrmutants were recovered, 89% of which were Pvd−and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10insertions were also found to be unstable in PAO1.

A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, lowmolecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15–SecY–adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M r value has been calculated to be 47100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis sec Y. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).

New shuttle vectors for Clostridium acetobutylicum were constructed, using as replicons the Gram-positive plasmid pIM13, and derivatives of the Gram-negative plasmid pBR322, including pUC19. These vectors transformed C. acetobutylicum at a high frequency (up to 106transformants per μg DNA) by PEG-mediated protoplast transformation. A mutant host strain, NI-4082, was isolated on the basis of its ability to maintain plasmid pIM13 stably in the absence of selection pressure. The shuttle vectors showed no segregational or structural instability in this mutant strain. Moreover, the results suggested a relationship between segregational instability and the multimerization of pIM13 in C. acetobutylicum. The host/vector system described possessed all the properties required for efficient gene cloning in this species.

The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.

Bacteriophage BFK20 was isolated from a Brevibacterium flavum strain that had become contaminated during industrial fermentation. BFK20 has a polyhedral head 50 nm wide and a non-contractile tail 200 nm long and 10 nm in diameter. The genome of this bacteriophage consists of a linear double stranded DNA molecule of 44–45 kb with cohesive ends. The capsid of phage BFK20 contains nine polypeptides with molecular masses from 22.0–108.0 kDa. BFK20 DNA was used as a donor for fragments carrying promoters and transcription-terminators.

A preliminary endonuclease restriction map of a bacteriophage isolated from Desulfovibrio vulgaris has been established. BamHI cleaved whole phage DNA into four fragments while HindIII cut the same DNA into seven fragments. Mapping studies succeeded in linking the four BamHI fragments into two DNA segments; however, no linkage between the two segments was detected. These data imply that two phages were induced from cultures of D. vulgaris and that the two segments represented the DNA from these phages. Support for this hypothesis came from size approximation of restriction enzyme fragments, electron micrographs, and density gradients.

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.

Auxotrophic mutants were isolated in two strains of Trichosporon cutaneum. Complementing auxotrophs were hybridized by protoplast fusion. Some of the fusants were apparently transient diploids and segregated to give recombinant marker combinations.

A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed β-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.

The production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.