- Volume 146, Issue 7, 2000

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 °C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 °C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 °C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1·6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

In contrast to what happens in Agrobacterium tumefaciens and Rhizobium meliloti, synthesis of periplasmic cyclic 1,2-β-glucan in Brucella spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-β-glucan synthetase of Brucella spp. was not inhibited by 0·5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The Brucella abortus cyclic 1,2-β-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-β-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0·25 M NaCl or 0·5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1,2-β-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-β-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that Brucella cyclic 1,2-β-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in Agrobacterium or Rhizobium backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-β-glucan synthesis in Brucella is due to two convergent mechanisms: a) the presence of a cyclic 1,2-β-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1,2-β-glucan synthetase.

Comamonas testosteroni TA441 degrades phenol by a meta-cleavage pathway after the occurrence of a spontaneous mutation that derepresses the aphKLMNOPQB operon encoding phenol hydroxylase and catechol 2,3-dioxygenase, the enzymes for the initial two steps of the degradation pathway. A gene cluster, aphCEFGHJI, encoding the meta-pathway enzymes for degradation of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates was found downstream of the aphK operon. The upstream operon and the downstream gene cluster were found to be separated by two open reading frames of unknown function and an oppositely oriented aphT gene, which is similar to regulatory genes for ortho-cleavage of catechol or chlorinated catechols. A promoter assay using an aphC::lacZ transcriptional fusion plasmid revealed that the aphC promoter activity is induced by both phenol and HMS. The phenol-dependent induction was mediated by AphR and the HMS-dependent induction was mediated by AphT. The aphC promoter in strain TA441 was not silenced, unlike the cases of the aphK and aphR promoters, and was highly induced by HMS.

Staphylococcus epidermidis can express three different cell-surface-associated proteins, designated SdrF, SdrG and SdrH, that contain serine-aspartate dipeptide repeats. Proteins SdrF and SdrG are similar in sequence and structural organization to the Sdr proteins of Staphylococcus aureus and comprise unique 625- and 548-residue A regions at their N termini, respectively, followed by 110–119-residue B-repeat regions and SD-repeat regions. The C termini contain LPXTG motifs and hydrophobic amino acid segments characteristic of surface proteins covalently anchored to peptidoglycan. In contrast, SdrH has a short 60-residue A region at its N terminus followed by a SD-repeat region, a unique 277-residue C region and a C-terminal hydrophobic segment. SdrH lacks a LPXTG motif. Recombinant proteins representing the A regions of SdrF, SdrG and SdrH were expressed and purified from Escherichia coli. Antisera specific to these proteins were raised in rabbits and used to identify Sdr proteins expressed by S. epidermidis. Only SdrF was released from lysostaphin-generated protoplasts of cells grown to late-exponential phase. SdrG and SdrH remained associated with the protoplast fraction and thus appear to be ineffectively sorted along the conventional pathway used for cell-wall-anchored proteins. In Southern hybridization analyses, the sdrG and sdrH genes were present in all 16 strains tested, whilst sdrF was present in 12 strains. Antisera from 16 patients who had recovered from S. epidermidis infections contained antibodies that reacted with recombinant A regions of SdrG and SdrH, suggesting that these proteins can be expressed during infection.

Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.

A regulon from Streptococcus mutans that plays a role in the utilization of β-glucosides has been isolated, sequenced and subjected to sequence analysis. This regulon encodes a β-glucoside-specific Enzyme II (EII) component (bglP) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a phospho-β-glucosidase (bglA) which is responsible for the breakdown of the phospho-β-glucosides within the cell. Both the bglP and bglA gene products have significant similarity with proteins that have similar functions from Clostridium longisporum, Listeria monocytogenes, Erwinia chrysanthemi, Escherichia coli, Klebsellia oxytoca and Bacillus subtilis. The potential functions of the BglP and BglA proteins are supported by phenotypic data from both S. mutans and E. coli. A chromosomal deletion in S. mutans spanning the bglP and bglA genes resulted in a strain that was unable to hydrolyse the β-glucoside aesculin in the presence of glucose. When glucose was removed from the medium, the deletion strain regained the ability to break down aesculin. These data suggest that S. mutans possesses an alternative mechanism from the one described in this report for breaking down β-glucosides. This second mechanism was repressed by glucose while the regulon described here was not. Complementation studies in E. coli CC118 also suggest a potential role for this regulon in the utilization of other β-glucosides. When a plasmid containing the 8 kb β-glucoside-specific regulon was transformed into E. coli CC118, the transformed strain was able to break down the β-glucoside arbutin.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is widely found among Gram-positive bacteria. It is the major source of carbohydrate transport in the dental pathogen Streptococcus mutans. The transported carbohydrates are fermented to produce large amounts of lactic acid which initiates dental caries. The authors have isolated the S. mutans gene for the mannitol-specific Enzyme II (EII) component of the PTS, mtlA, and the adjacent mtlR gene, which is located in the same operon. The mtlR gene is located between mtlA and the genes mtlF and mtlD. The nucleotide sequence of the mtlA and mtlR loci has been determined. The deduced mtlA gene product of S. mutans consists of 589 amino acids with a molecular mass of 62·0 kDa. It exhibits similarity with the mtlA gene products from other organisms. However, the similarity between these proteins is generally restricted to the 470 amino-terminal residues of the S. mutans protein. This region would correspond to the EIICB domains of the PTS. The authors have previously shown that the S. mutans mtlF gene product exhibits 76·6% similarity to the carboxyl-terminal 143 amino acids of the Escherichia coli mtlA product and that the mtlF gene encodes the EIIA domain of the PTS. Thus, the genes that encode the EIICB and the EIIA domains are separated by approximately 2250 bp. In many organisms, all of the EII domains may be fused together to form one molecule. The fact that these domains are separated by this distance in S. mutans supports the hypothesis that various functional domains of the PTS have been rearranged during evolution. The sequence of the 119 carboxyl-terminal amino acids of the S. mutans mtlA gene product also displays homology to the carboxyl-terminal end of the EIIB domain of various mannitol PTSs. Thus, this domain may have been duplicated in S. mutans during evolution of the operon. The mtlR gene is located in the same operon structure as mtlA but these loci are separated by an intragenic space. The precise 5′ end of the mtlR locus cannot be determined either by in vitro transcription–translation assays or based upon nucleotide sequence analysis because of the apparent lack of a ribosome-binding site preceding the gene. The deduced mtlR gene product, which consists of approximately 650 amino acids with a molecular mass of 75·3 kDa, exhibits limited similarity to several potential transcriptional regulators. However, the exact function of this locus is currently unknown.

Essential two-component systems are now being identified in bacteria. The Bacillus subtilis yycF gene encoding a response regulator, and its orthologue in Staphylococcus aureus, were reported recently to be essential for cell growth, although genes under their control have yet to be identified. The essential nature of the yycF regulator gene and its cognate kinase gene, yycG, in B. subtilis was also noted during the course of construction of a knockout mutant bank of newly identified genes in the genome sequence project. It was found that yycG could be deleted in the presence of an active form of the YycF protein, thereby suggesting direct interaction between YycG and YycF. Production of mini-cells and reduction in cell length occurred when the YycF regulator was overproduced in B. subtilis. These observations led to the finding that YycF overproduction up-regulated the expression from the P1 promoter of the cell division operon, ftsAZ. In addition, the YycF protein binds to the P1 promoter region in vitro. These results clearly indicate that the essential two-component regulatory system encoded by yycF and yycG genes has the potential to modulate expression of the ftsAZ operon in B. subtilis.

The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.

Conservation of the sporulation processes between Bacillus spp. and Clostridium spp. was investigated through evolutionary and complementation analyses of σE. Alignment of partial predicted σE amino acid sequences from three Bacillus spp., Paenibacillus polymyxa and five Clostridium spp. revealed that amino acid residues previously reported to be involved in promoter utilization (M124, E119 and N120) and strand opening (C117) are conserved among all these species. Phylogenetic analyses of various sigma factor sequences from endospore-forming bacteria revealed that homologues of σE, σK and σG clustered together regardless of genus, suggesting a common origin of sporulation sigma factors. The functional equivalence between Clostridium acetobutylicum σE and Bacillus subtilis σE was investigated by complementing a non-polar B. subtilis σE null mutant with the spoIIG operon from either B. subtilis (spoIIG Bs) or C. acetobutylicum (spoIIG Ca). Single-copy integration of spoIIG Bs into the amyE locus of the σE null mutant completely restored the wild-type sporulation phenotype, while spoIIG Ca only partially restored sporulation. Maximal expression of spoIIG Ca–lacZ occurred approximately 12 h later than maximal expression of spoIIG Bs–lacZ. Differences in temporal expression patterns for spoIIG Ca and spoIIG Bs in the B. subtilis background may at least partially explain the observed sporulation complementation phenotypes. This study suggests a common phylogenetic ancestor for σE in Bacillus spp. and Clostridium spp., although regulation of σE expression may differ in these two genera.

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70% identity to ClpA of Rhodobacter blasticus, was identified and sequenced. Following heterologous expression in Escherichia coli strains SG1126 (ΔclpA) and SG1127 (Δlon ΔclpA), b-ClpA replaced the function of E. coli ClpA, participating in the degradation of abnormal proteins. A b-clpA null mutant of B. suis was constructed, and growth experiments at 37 and 42 °C showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 °C. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B. suis overexpressing b-clpA behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of B. suis. These results showed that in B. suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.

Azotobacter vinelandii forms both catecholate and azotobactin siderophores during iron-limited growth. Azotobactin is repressed by about 3 μM iron, but catecholate siderophore synthesis continues up to a maximum of 10 μM iron. This suggests that catecholate siderophore synthesis is regulated by other factors in addition to the ferric uptake repressor (Fur). In this study the first gene required for catecholate siderophore biosynthesis, which encodes an isochorismate synthase (csbC), was isolated. The region upstream of csbC contained a typical σ70 promoter, with an iron-box overlapping the −35 sequence and a Sox-box (Box 1) overlapping the −10 sequence. Another Sox-box was found further upstream of the −35 sequence (Box 2). Also upstream, an unidentified gene (orfA) was detected which would be transcribed from a divergent promoter, also controlled by an iron-box. The activity of csbC and a csbC::luxAB fusion was negatively regulated by iron availability and upregulated by increased aeration and by superoxide stress. The iron-box in the csbC promoter was 74% identical to the Fur-binding consensus sequence and bound the Fur protein of Escherichia coli with relatively high affinity. Both Box 1 and Box 2 were in good agreement with the consensus sequence for binding the SoxS protein of E. coli and Box 1 was in very good agreement with the Sox-box found in the fpr promoter of A. vinelandii, which is also regulated by superoxide stress. Both Sox-boxes bound a protein found in A. vinelandii cell extracts, with Box 1 exhibiting the higher binding affinity. The Sox protein identified in this assay appeared to be constitutive, rather than inducible by superoxide stress. This indicates that the Sox response in A. vinelandii is different from that in E. coli. These data support the hypothesis that catecholate siderophore biosynthesis is under dual control, repressed by a Fur–iron complex and activated by another DNA-binding protein in response to superoxide stress. The interaction between these regulators is likely to account for the delay in ferric repression of catecholate siderophore production, since these siderophores have an additional role to play in the protection of iron-limited cells against oxidative damage.

The authors report the isolation and molecular characterization of a bacteriophage, ϕCPG1, which infects Chlamydia psittaci strain Guinea pig Inclusion Conjunctivitis. Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the ϕX174 family of bacteriophages. The single-stranded circular DNA genome of ϕCPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described Chlamydia bacteriophage (Chp1) that infects avian C. psittaci. Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees. Lesser sequence similarities were seen between two ORF products and the major capsid protein of the ϕX174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids. Phage ϕCPG1 is the second member of the genus Chlamydiamicrovirus, the first to infect a member of a Chlamydia species infecting mammals. Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae. This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of ϕCPG1. This finding suggests that C. pneumoniae has been infected by a phage related to ϕCPG1 and that infection resulted in integration of some of the phage genome into the C. pneumoniae genome.