Bacteriocin production inAgrobacterium tumefaciens

Agrobacterium tumefaciens C58 forms “plaques” during layer cultivation. The “plaques” were shown not to be caused by the presence of a temperate bacteriophage or by random contamination. The “plaques” and their central microcolonies were used to repeatedly isolate cultures producing an antibiotic substance against the original strainA. tumefaciens C58, other nopaline strains, some octopine strains ofA. tumefaciens and some strains of the related genusRhizobium. The substance is thus a bacteriocin; in analogy to agrocins 84 and D286 it was named agrocin C58. The agrocin is not inactivated by trypsin. Its production by strain C58 was found only on cultivation on solid but not liquid media. The producing isolate ofA. tumefaciens C58 (strain C58i2) contains neither plasmid pTiC58 nor the plasmid analogous to pAgK84 which controls the production of agrocin 84 inA. radiobacter K84.     

Isolation and properties of transfer regulatory mutants of the nopaline Ti-plasmid pTiC58

Summary Conjugal transfer of the nopaline Ti-plasmid pTiC58 is inducible by the phosphorylated opines, agrocinopines A and B. Although the uninduced level of transfer is negligible (< 10^–7 per donor), some transconjugants have been isolated from crosses performed in the absence of agrocinopine. These transconjugants harbour mutant Ti-plasmids that transfer constitutively (>10^–3 per donor). These mutants have several other correlated phenotypes including constitutive uptake of agrocinopine A, supersensitivity to agrocin 84 and the ability to prevent the excretion of agrocin 84 when they are present in the same cell as the agrocin 84 biosynthetic plasmid pAt-84a.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Sensitivity of differentAgrobacterium strains to agrocin 84

Sensitivity of fourteenA. tumefaciens andA. rhizogenes strains to agrocin 84 was followed. A new agrocin 84 sensitivity of mannopine strains ofA. rhizogenes 8196 and TR101 was described. Agropine strains ofA. rhizogenes andA. tumefaciens were sensitive to agrocin 84, too. Agrocin 84 sensitivity of succinamopine and mannopine strains ofA. tumefaciena was also confirmed.     

Characterization of the acc operon from the nopaline-type Ti plasmid pTiC58, which encodes utilization of agrocinopines A and B and susceptibility to agrocin 84.

The acc locus from the Ti plasmid pTiC58 confers utilization of and chemotaxis toward agrocinopines A and B (A+B), as well as susceptibility to a highly specific antiagrobacterial antibiotic, agrocin 84. DNA sequence analyses revealed that acc is composed of eight open reading frames, accR and accA through accG. Previous work showed that accR encodes the repressor which regulates this locus, and accA codes for the periplasmic binding protein of the agrocinopine transport system (S. Beck Von Bodman, G. T. Hayman, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 89:643-647, 1992; G. T. Hayman, S. Beck Von Bodman, H. Kim, P. Jiang, and S. K. Farrand, J. Bacteriol. 175:5575-5584, 1993). The predicted proteins from accA through accE, as a group, have homology to proteins that belong to the ABC-type transport system superfamily. The predicted product of accF is related to UgpQ of Escherichia coli, which is a glycerophosphoryl diester phosphodiesterase, and also to agrocinopine synthase coded for by acs located on the T-DNA. The translated product of accG is related to myoinositol 1 (or 4) monophosphatases from various eucaryotes. Analyses of insertion mutations showed that accA through accE are required for transport of both agrocin 84 and agrocinopines A+B, while accF and accG are required for utilization of the opines as the sole source of carbon. Mutations in accF or accG did not abolish transport of agrocin 84, although we observed slower removal of the antibiotic from the medium by the accF mutant compared to the wild type. However, the insertion mutation in accF abolished detectable uptake of agrocinopines A+B. A mutation in accG had no effect on transport of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This finding suggests that agrocin 84 is activated by AccF after being transported into the bacterial cell.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocinopine-type opines

Summary Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.     

Occurrence of free and conjugated 12,13-epoxytrichothecenes and zearalenone in banana fruits infected with Fusarium moniliforme.

Agrobacterium tumefaciens J73, a biotype 2 strain harboring a nopaline Ti plasmid, was found to produce an agrocin active against a broad range of A. tumefaciens strains, including grapevine isolates. Sensitivity to J73 is not encoded by a Ti plasmid. Optimal conditions for the production of the agrocin were determined.     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.Penicillin binding proteins (PBPs) are critical components of the cell wall synthesis machinery in bacteria. These membrane-associated proteins are broadly classified as low-molecular-mass (LMM) PBPs that are monofunctional d,d-carboxypeptidase enzymes or multimodular high-molecular-mass (HMM) PBPs with multiple functional roles. PBPs, in general, are anchored to the cytoplasmic membrane by a noncleavable pseudo-signal peptide. In the case of the HMM PBPs, the cytoplasmic C-terminal domain binds penicillin and catalyzes peptidoglycan cross-linking, whereas the juxtamembrane N-terminal domain participates in transglycosylation (12). The catalytic penicillin-binding (PB) module also occurs as part of penicillin sensor transducers, such as Staphylococcus aureus MecR and Bacillus licheniformis BlaR (15). The transpeptidase activity in HMM PBPs is based on a conserved lysine residue located in the so-called catalytic S-X-X-K motif, whereas the other conserved S-X-N and K(H)-T(S)-G motifs govern carboxypeptidase activity and bind penicillin (20). The carboxypeptidase domain of PBPs is the target for β-lactam antibiotics in susceptible staphylococci (with penicillin MICs as low as 1 μg/ml).The transpeptidase activity of the PBPs occurs at the d-Ala-d-Ala terminus of a precursor disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Ala-l-Lys-d-Ala-d-Ala. This reaction is initiated by acylation involving a nucleophilic attack by the active-site serine on the penultimate d-Ala residue to form an acyl-enzyme complex. The C-terminal d-Ala is subsequently released from the peptide chain, followed by deacylation. In the case of HMM PBPs, deacylation occurs when an amino group on a second peptide substrate acts as an acceptor, resulting in a peptide cross-link between two adjacent peptidoglycan strands. The carboxypeptidase activity of LMM PBPs follows a similar reaction scheme, except that the acceptor in this case is a water molecule. β-Lactam antibiotics mimic the substrates of the PBPs. However, unlike the natural substrate, the β-lactam-PBP acyl adduct is stable and results in irreversible inhibition of PBP function. The β-lactam-PBP acyl adduct has been characterized extensively, with over 50 protein-antibiotic complexes reported to date (37). Thus, in contrast to the nonessential LMM PBPs, HMM PBPs constitute lethal targets for β-lactam antibiotics (6).Staphylococcus aureus is a gram-positive coccus and is one of the leading causes of high morbidity and mortality associated with both community- and hospital-associated infections (42, 46). This coccus shows extensive genomic variation, with over 22% of the genome dedicated to dispensable regions. A genome-scale analysis of a clinical strain of S. aureus is of particular interest in this context, wherein the conversion of a susceptible strain of S. aureus to a multidrug-resistant phenotype was shown to involve just 35 mutations in 13 loci, achieved within 3 months (36). Of the five PBPs in S. aureus, an acquired PBP, PBP2a, is the most extensively examined, as it was noted to be a specific marker for methicillin-resistant S. aureus (MRSA) strains. Among the intrinsic PBPs, PBP1 has been shown to play a key role in cell growth and division (2). PBP2 is a dual-function enzyme with both transglycosylase and transpeptidase activities, and inhibition of this protein leads to restrained peptidoglycan elongation and subsequent leakage of cytoplasmic contents due to cell lysis (34, 40). Inactivation of PBP3 neither changes the muropeptide composition of the cell wall nor significantly decreases the rate of autolysis. However, cells of abnormal size and shape and with disoriented septa are produced when bacteria with inactivated PBP3 are grown with sub-MIC levels of methicillin (29).S. aureus PBP4 is a carboxypeptidase and is needed for the secondary cross-linking of peptidoglycan (19). However, it is not essential for cell growth under laboratory conditions, because mutants of S. aureus defective in PBP4 are viable (48). Overexpression of PBP4 was noted to result in an increase in β-lactam resistance and in greater cross-linking of the peptidoglycan (18). S. aureus PBP4 is similar to other LMM PBPs and is grouped within the superfamily of penicillin-susceptible and penicillin-interacting enzymes. However, homologues of PBP4 have a different phenotype in other species (1, 15). For example, a mutation of PBP4 in Pseudomonas aeruginosa triggers an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC. The P. aeruginosa PBP4 mutant also activates CreBC, a two-component regulator, thereby mediating β-lactam resistance (33). Indeed, S. aureus PBP4 has been suggested to have different functions in strains with different genetic backgrounds (26). However, based on in vitro and genetic data, S. aureus PBP4 is primarily a transpeptidase and has little d,d-carboxypeptidase activity. This is also supported by the observation that increased carboxypeptidase activity decreases cell wall cross-linking due to loss of the free d-Ala-d-Ala termini necessary for transpeptidation (10). In this context, it is pertinent that pbp4 gene knockout strains of S. aureus were more resistant to the glycopeptide antibiotic vancomycin (46).Here we present the biochemical and structural characteristics of PBP4 from S. aureus strain COL. S. aureus PBP4 is a β-lactamase. A comparison of the crystal structure of S. aureus PBP4 in complex with antibiotic with that of its Escherichia coli homologue, PBP5, provides a conformational and biochemical rationale for the β-lactamase activity of PBP4. Monitoring the expression of PBP4 in the MRSA strain COL and representative clinical strains of S. aureus suggested that the expression level of PBP4 does not fluctuate substantially across these strains. Together, these data on the structure, expression, activity, and regulation of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.     

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.     

Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.     

Plasmid Heterogeneity in Spanish Isolates of Agrobacterium tumefaciens from Thirteen Different Hosts

Plasmid DNA was isolated from 80 Spanish isolates of Agrobacterium tumefaciens from 13 hosts of several geographical and temporal origins. One to five plasmids occurred in all of the isolates studied. Plasmid sizes varied between 5 and greater than 1,000 MDa. Generally, there was no correlation between plasmid number or size and geographical origin, host, biovar, sensitivity to agrocin 84, or opine-catabolizing ability of the different isolates.     

Acquisition of Five High-Mr Penicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from Streptococcus mitis to Streptococcus pneumoniae

Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level β-lactam resistance by interspecies gene transfer.     

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N₂, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.