- Volume 145, Issue 4, 1999

Summary: The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. Immunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of lytM expression in two global regulatory mutants, agr and sar, showed that expression of lytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X11-Val-X12/20-Gly-X5–6-His. Atomic absorption spectrometric analysis of LytM revealed the presence of 0·9 mol zinc (mol LytM)-1.

Summary: Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.

Summary: Southern blotting, sequence analysis and PCR experiments showed that Mycobacterium bovis and Mycobacterium bovis BCG lack a 12·7 kb fragment present in the genome of Mycobacterium tuberculosis. This region is 337 bp downstream of the RD2 region, which was previously described as being absent from some M. bovis BCG strains. The 12·7 kb fragment should be useful as a target for a PCR test to differentiate M. tuberculosis and M. bovis. An analysis of the 12·7 kb region suggests that it represents a deletion in M. bovis rather than an insertion in M. tuberculosis. The deletion removes most of the mce-3 operon, one of four highly related operons which may be involved in cell entry, and therefore it may contribute to differences in virulence or host range in the two species.

Summary: Haemophilus influenzae binds haemoglobin and the haemoglobin-haptoglobin complex and utilizes either as a sole source of haem. Previously, a DNA fragment was cloned from H. influenzae that encodes an approximately 120 kDa protein (HgpA) expressing haemoglobin-binding activity in Escherichia coli. Partial sequence analysis revealed significant homology of HgpA with other bacterial haem- and iron-utilization proteins, and a length of CCAA repeating units immediately following the nucleotide sequence encoding the putative leader peptide. In the present study, the complete nucleotide sequence of the cloned DNA fragment was determined and the sequence was analysed. In addition to homology with other haem- and iron-utilization proteins, seven regions typical of TonB-dependent proteins were identified. The transcript of hgpA was determined to be monocistronic by RT-PCR. PCR performed with different colonies of a single H. influenzae strain at one CCAA-repeat-containing locus indicated varying lengths of CCAA repeats, suggesting that haemoglobin and haemoglobin-haptoglobin binding in H. influenzae is regulated by strand slippage across CCAA repeats, as well as by haem repression. E. coli containing cloned hgpA bound both haemoglobin and the haemoglobin-haptoglobin complex. A deletion/insertion mutation of hgpA was constructed in H. influenzae strain HI689. Mutation of hgpA did not affect the ability of H. influenzae either to bind or to utilize haemoglobin or haemoglobin-haptoglobin following growth in haem-deplete media. Affinity purification of haemoglobin-binding proteins from the mutant strain revealed loss of the 120 kDa protein and an increased amount of a 115 kDa protein, suggesting that at least one additional haemoglobin-binding protein exists.

Summary: Five rRNA operons (rrn) were found by hybridization in the genome of ‘Brevibacterium lactofermentum’ ATCC 13869 and Corynebacterium glutamicum ATCC 13032. ‘B. lactofermentum’ DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-55 rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that ‘B. lactofermentum’ is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5′ end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

Summary: A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chiC, with the same transcriptional direction, and two unlinked genes, chiP and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, ChiB and ChiC exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while ChiP and ChiQ belonged to family 20. ChiP and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; ChiB bound chitin but had no chitinase activity; ChiC possessed strong chitinase activity and also bound chitin. Production of ChiC in S91 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of ChiC proteins in a chitinase-negative mutant. In Escherichia coli, ChiC appeared to be expressed from its own promoter.

Summary: Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine. The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine. Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases. Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival. This is believed to be the first report of a hmgA-homologous gene in bacteria.

Summary: The authors have examined the response to UV-A irradiation of the UV-A-resistant marine cyanobacterium Oscillatoria sp. NKBG 091600, which produces the UV-A-absorbing compound biopterin glucoside. The expression of a 60 kDa protein was markedly induced at 500 min after UV-A irradiation. This protein was identified by N-terminal amino acid sequence analysis as GroEL. Northern blot analysis demonstrated that GroEL synthesis was controlled by UV-A at the transcriptional level. A CIRCE element and a putative SOS consensus sequence were found upstream of the groESL operon, overlapping two putative promoter sequences. Primer extension analysis revealed that groESL transcription in UV-A-induced cells starts from the proximal promoter overlapped by the SOS consensus sequences. This indicates that an SOS response regulation is instrumental in UV-A-induced GroEL expression of Oscillatoria sp. NKBG 091600. Furthermore, this UV-A-inducible GroEL may function to upregulate biopterin glucoside biosynthesis, thereby allowing growth under UV-A irradiation.

Summary: Bacillus subtilis responds to various stimuli (heat, ethanol and salt stress, energy starvation) with the induction of general stress proteins (GSPs). Most of them belong to the stress and stationary-phase regulon controlled by the alternative sigma factor σB. The majority of σB-dependent proteins are thought to provide a precautionary general stress resistance in stressed or starved cells. In this report, the identification and transcriptional analysis of nine new members of the σB regulon are described. The biochemical function was not determined for any of the proteins encoded by the nine new σB-dependent stress genes, however, similarities to proteins in the databases allowed a distinction between proteins with putative (i-iv) and unknown (v) function. The putative functions of BmrU, YcdF, YdaD, YdaP, YhdN and YocK underline the suggested protective role of σB-dependent GSPs and also elucidate new areas where σB might play an important role. (i) The finding that the bmrUR operon is under σB control indicates that the elimination of multidrug compounds might be a new function in multiple stress resistance. (ii) YcdF and YdaD resemble NAD(P)-dependent dehydrogenases. Both proteins could be involved in the generation of NAD(P)H and therefore in the maintenance of the intracellular redox balance under stress. (iii) The ydaP gene might belong to the increasing number of σB-dependent genes whose orthologues are under the control of σS in Escherichia coli, indicating that both regulons may fulfil similar functions. (iv) YhdN shows weak similarities to potassium ion channel proteins and YocK shows resemblance to the DnaK suppressor protein DksA. (v) Three new σB-dependent genes (ydaE, ydaG and yfkM) encoding proteins with still unknown functions were also described. Further analyses of corresponding mutants might allow a first prediction of their function within the framework of the general stress regulon.

Summary: The genome sequence of Mycobacterium tuberculosis H37Rv was found to contain 56 loci with homology to insertion sequences (ISs). As well as the previously described IS6110, IS1081, IS1547 and IS-like elements, new ISs belonging to the IS3, IS5, IS21, IS30, IS110, IS256 and ISL3 families were identified. In addition, six ISs created a grouping of their own to form a new family (the IS1535 family). Elements with similarity to ISs in other actinomycetes were identified, suggesting the movement of ISs between related genera. The location of ISs on the chromosome revealed that an approximately 600 kb region close to the origin of replication lacks ISs, pointing to the possible detrimental effect of insertions in this area. Analysis of the distribution of ISs through the tubercle strains Mycobacterium africanum, M. microti, M. bovis, M. bovis BCG Pasteur, M. tuberculosis H37Ra, M. tuberculosis CSU#93 and 29 clinical isolates revealed that only IS1532, IS1533, IS1534, and IS1561 were absent from some of the strains tested. A novel repeated sequence, the REP13E12 family, is described that is present in seven copies on the M. tuberculosis H37Rv chromosome and which contains a probable phage attachment site. This study therefore offers an insight into the possible role of ISs and repetitive elements in the evolution of the M. tuberculosis genome, as well as identifying genetic markers that may be useful for phylogenetic and epidemiological analysis of the tubercle complex.

Summary: This study investigated the ability of anaerobic periodontal bacteria to inactivate and resist killing by antimicrobial peptides through production of extracellular proteases. Antibacterial activities of peptides were assessed in a double-layer agarose diffusion assay, and MICs and MBCs were determined in broth microdilution assays. Culture supernates of Porphyromonas gingivalis and Prevotella spp. inactivated mastoparan, magainin II and cecropin B whilst Gram-positive oral supragingival bacteria had no effect. Inactivation was prevented by protease inhibitors and was unaffected by 45% human serum. Purified proteases from the periodontopathogen Porph. gingivalis inactivated peptides [cecropin B, brevinin, CAMEL (cecropin A 1–7 + melittin 2–9), mastoparan] as would be predicted from the amino acid sequences of the peptides and the known bond specificities of these Arg-x and Lys-x enzymes. MALDI-TOF MS revealed that inactivation of cecropin B by Porph. gingivalis protease was due to specific cleavage of the molecule. Inactivation of cecropin B by proteases took 10–15 min. Paradoxically, MICs of cecropin B against Proph. gingivalis and Prevotella intermedia were low, while Prevotella nigrescens was resistant, suggesting that production of proteases alone is insufficient to protect Proph. gingivalis and Prev. intermedia from the action of antimicrobial peptides. Thus, antimicrobial peptides could be developed as therapeutic agents targeted against specific periodontal pathogens.

Summary: Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and Δ1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0·93 nkat (mg protein)-1 and 4·3 nkat (mg protein)-1, respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28·9 kDa as calculated from the sequence. Apparent K m values for PCA and NADH were 0·19 mM and 0·025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent K m values: 1·55 mM for proline and 10·5 mM for NAD at pH 10·0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

Summary: Continuous cultures of Chromatium vinosum DSM 185 were shifted from a high to a low irradiance (67 to 4 μE m-2 s-1) and vice versa (4 to 67 μE m-2 s-1). The kinetics of photoacclimation of the cultures were analysed during these transitions until steady state was reached. When irradiance was shifted from 4 to 67 μE m-2 s-1, bacteriochlorophyll synthesis halted for 4 h. During this period, pigments were progressively diluted in the newly formed biomass, resulting in a lower specific pigment content. The specific growth rate of the organisms did not change immediately after the shift, but rather underwent a gradual increase during the following 10 h. This transition was accompanied by a transient increase in the levels of glycogen, indicating that CO2 fixation rates increased immediately after the shift, and that unused photosynthate was stored as glycogen. The shift from a high to a low irradiance was characterized by an immediate drop in the specific growth rate to virtually zero, and by comparatively sharp decreases in the specific rates of sulfur and sulfide oxidation and in the specific rate of glycogen accumulation. The specific content of bacteriochlorophyll a increased during the first 10 h. During the same period the specific content of glycogen decreased.